Modified MRNA vaccines encoding herpes simplex virus glycoproteins and uses thereof

ABSTRACT

Compositions for the prevention and treatment of genital herpes, comprising nucleoside modified mRNAs that encode herpes simplex virus (HSV) glycoproteins, including those involved in virus entry and immune evasion, and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/640,008, filed Feb. 18, 2020, which is a National Phase of PCTInternational Application No. PCT/IB2018/056210 filed Aug. 17, 2018,claiming the benefit of U.S. Patent Application No. 62/546,648, filedAug. 17, 2017 and U.S. Patent Application No. 62/701,019, filed Jul. 20,2018 which are hereby incorporated by reference.

GOVERNMENT INTEREST STATEMENT

This invention was made with government support under AI104854 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on May 30, 2021, isnamed P-572137-US1 ST25.txt and is 57.2 kilobytes in size.

FIELD OF INVENTION

The present invention provides compositions for the prevention andtreatment of genital herpes, comprising nucleoside modified mRNAs thatencode herpes simplex virus (HSV) glycoproteins, including thoseinvolved in virus entry and immune evasion, and methods of use thereof.

BACKGROUND OF THE INVENTION

A genital herpes vaccine is urgently needed to prevent pain andsuffering, reduce the incidence of neonatal herpes, and decrease therisk of HIV acquisition and transmission that accompanies genitalinfection. Approximately a half-billion people worldwide are infectedwith herpes simplex virus type 2 (HSV-2), the virus that causes genitalherpes. In some individuals, infection results in painful, recurrentgenital ulcers, while in others, the infection remains quiescent. Inboth settings, infected individuals may transmit virus to their intimatepartners. Genital herpes increases the risk that an infected person willacquire HIV if exposed during sexual intercourse. A vaccine for genitalherpes is urgently needed, yet none is available.

Chiron Corp. evaluated a prophylactic vaccine containing two HSV-2glycoproteins involved in virus entry, glycoproteins B (gB2) and D (gD2)given with MF59 as adjuvant. The vaccine did not protect seronegativepartners from HSV-2 infection, although it delayed onset of infectionover the first 5 months after immunization. GlaxoSmithKline (GSK)assessed a prophylactic vaccine using gD2 antigen with monophosphoryllipid A (MPL) and alum as adjuvants. Overall, no protection againstgenital lesions was detected, although significant protection was notedin a subgroup of HSV-1 and HSV-2 doubly seronegative women. A follow-uptrial was performed in doubly seronegative women that showed no overallprotection against genital herpes; however, the vaccine was efficaciousagainst HSV-1. This result was noteworthy because HSV-1 accounted for60% of genital herpes infections in the control group. These studiesindicate that targeting a vaccine to block HSV-2 entry is notsufficient.

HSV-1 and HSV-2 gC are immune evasion molecules that function asregulators of the complement cascade. During complement activation, C3,the most abundant complement protein, is cleaved to C3b, which activatesthe membrane attack complex leading to virus neutralization and lysis ofinfected cells. C3b stimulates B- and T-cell responses and serves as alink between innate and acquired immunity. HSV-1 and HSV-2 gC bind C3bto inhibit activities mediated by C3b. Immunization with gC1 and gC2produces antibodies that bind to the glycoprotein and block its immuneevasion functions.

HSV-1 and HSV-2 glycoprotein E (gE) function as immune evasion moleculesby binding the Fc domain of an IgG molecule that is bound by its F(ab′)2domain to its target. A vaccine containing gE2 subunit antigen producesantibodies that bind to gE2 and block its immune evasion functions.HSV-2 gC2 and gE2 perform activities similar to mammalian complement andIgG Fc regulatory proteins, yet share no sequence homology withmammalian receptors, which suggests virtually no risk that immunizationwill induce autoimmunity.

Previous work from our lab examined vaccines containing gC, gD and gEand found that such vaccines provide protection against HSV infection.However, it is not known if mRNA vaccines encoding HSV gC, gD and gEwould be effective in protecting against HSV infection.

Using nucleic acids as vaccines has multiple advantages. Nucleic acidvaccines can induce both humoral and cellular immune responses; have loweffective dosages; are simple to manipulate; avail rapid testing; arecost-effective and reproducible in large scale production and isolation;can be produced at high frequency and are easily isolated; are moretemperature-stable than conventional vaccines; have a long shelf-life;are easy to store and transport; and are unlikely to require a coldchain (Shedlock & Weiner, J Leukocyte Biol. Vol 68, 2000).

In principle, either exogenous DNA or RNA can express proteins in themammalian body. Whether or not similar immune activity can be producedwith both DNA and mRNA expressed proteins is uncertain. Conventionalwisdom is that DNA is superior for the creation of vaccines and genetherapy due to its stability and ease of use.

DNA has been used in vaccines with success. DNA is fairly stable andunreactive and can be stored long term. However, DNA is self-replicatingand can be easily damaged by ultra-violet radiation. DNA based vaccinesmay also raise safety concerns due to possible insertion of DNA into thegenome, possible interruption of genes and formation of anti-DNAantibodies.

RNA vaccines exhibit important safety features. RNA is more reactivethan DNA and less stable but is resistant to ultra-violet radiation.mRNA does not integrate into the host chromosomes. The delivery of mRNAresults in faster expression of the antigen of interest and requiresfewer copies for expression. mRNA expression is transient, which adds toits safety. mRNA is more effective than DNA for protein production inpost mitotic and non-dividing cells because DNA requires translocationthrough the nuclear member and plasma membrane, while mRNA requirestranslocation only through the plasma membrane. mRNA is not only atemplate for translation, but also acts as a ligand for toll-likereceptors and is nuclease sensitive; therefore it presents less concernfor horizontal transmission.

In addition, RNA vaccines elegantly integrate adjuvanticity and antigenexpression, thereby mimicking relevant aspects of viral infections. Thisincreases their efficacy compared to inactivated vaccines that requirethe use of adjuvants, simplifying handling and production. RNA canaddress a range of dedicated immunologic pattern recognition receptors,including toll-like receptors 3, 7, and 8, RIG-I, MDA5, PKR, and othersthat may act synergistically and serve to enhance the induction ofantigen-specific adaptive B and T cell responses. Importantly, byantigen synthesis in transfected host cells, mRNA vaccines directlyintroduce antigen into cellular antigen processing and presentationpathways, granting access to MHC molecules and triggering T cellresponses, irrespective of the hosts MHC haplotype. This enables theinduction of polyclonal T cell responses that may act synergisticallywith other immune responses, including B cells. Also, endogenousproduction of antigen ensures faithful posttranslational modification(e.g. proteolytic processing, glycosylation, etc.) that may positivelyimpact immunogenicity.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides compositionscomprising one or more nucleoside modified mRNAs, wherein each of saidnucleoside modified mRNAs encodes a Herpes Simplex Virus (HSV)glycoprotein or immunogenic fragment thereof, and wherein saidnucleoside modified mRNA comprises one or more pseudouridine residues.

In another embodiment, the present invention provides compositionscomprising one or more nucleoside modified mRNAs, wherein each of saidmodified mRNAs encodes a Herpes Simplex Virus (HSV) glycoprotein orimmunogenic fragment thereof, and wherein said nucleoside modified mRNAcomprises 1-methylpseudouridine, wherein said pseudouridine residuescomprise m¹acp³Ψ (1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, m¹Ψ(1-methylpseudouridine), Ψm (2′-O-methylpseudouridine, m⁵D(5-methyldihydrouridine), m³Ψ (3-methylpseudouridine), or anycombination thereof.

In another embodiment, the present invention provides compositionscomprising modified mRNAs comprising one or more pseudouridine residues,wherein each of said modified mRNAs encode an a) HSV glycoprotein D (gD)or an immunogenic fragment thereof, b) HSV glycoprotein C (gC) or animmunogenic fragment thereof, c) HSV glycoprotein E (gE) or animmunogenic fragment thereof, or any combination thereof.

In another embodiment, the present invention provides a method oftreating a Herpes Simplex Virus (HSV) infection in a subject, the methodcomprising the step of administering to said subject a compositioncomprising one or more modified mRNAs, wherein each of said modifiedmRNAs encodes an HSV glycoprotein or immunogenic fragment thereof, andwherein said modified mRNA comprises pseudouridine residues.

In another embodiment, the present invention provides a method ofinducing an immune response in a subject, comprising the step ofadministering to said subject a composition comprising one or moremodified mRNAs, wherein each of said modified mRNAs encodes an HSVglycoprotein or immunogenic fragment thereof, and wherein said modifiedmRNA comprises pseudouridine residues.

In a further embodiment, the present invention provides a method ofsuppressing, inhibiting, or reducing the incidence of a Herpes SimplexVirus (HSV) infection in a subject, the method comprising the step ofadministering to said subject a composition comprising one or moremodified mRNAs, wherein each of said modified mRNAs encodes an HSVglycoprotein or immunogenic fragment thereof, and wherein said modifiedmRNA comprises pseudouridine residues.

In yet another embodiment, the present invention provides a method oftreating a Herpes Simplex Virus (HSV) infection in a subject, the methodcomprising the step of administering to said subject a compositioncomprising one or more modified mRNAs encoding a) an HSV glycoprotein D(gD) or an immunogenic fragment thereof, b) an HSV glycoprotein C (gC)or an immunogenic fragment thereof, c) an HSV glycoprotein E (gE) or animmunogenic fragment thereof, or any combination thereof.

In a yet a further embodiment, the present invention provides a methodof suppressing, inhibiting, or reducing the incidence of a HerpesSimplex Virus (HSV) infection in a subject, the method comprising thestep of administering to said subject a composition comprising one tothree modified mRNAs, wherein each of said modified mRNAs encodes a) HSVglycoprotein D (gD) or an immunogenic fragment thereof, b) HSVglycoprotein C (gC) or an immunogenic fragment thereof, and c) HSVglycoprotein E (gE) or an immunogenic fragment thereof, or anycombination thereof.

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C. Characterization of the translational product of theectodomain of gC2-, gD2- and gE2-modified mRNA in Vero cells.

FIG. 1A. Western blot showing expression of gC2 by modified mRNA.

FIG. 1B. Western blot showing expression of gD2 by modified mRNA.

FIG. 1C. Western blot showing expression of gE2 by modified mRNA.

FIG. 2A. gC2 antibody (Ab) response as determined by antigen-specificELISA in mice immunized with gD2 mRNA; or gC2 mRNA, gD2 mRNA and gE2mRNA each given at a different intradermal site (Trivalent-I); or gC2mRNA, gD2 mRNA and gE2 mRNA given in combination (Trivalent-C). Iindicates first immunization; II indicates second immunization.

FIG. 2B. gD2 Ab response as determined by antigen-specific ELISA in miceimmunized with gD2 mRNA; or gC2 mRNA, gD2 mRNA and gE2 mRNA each givenat a different intradermal site (Trivalent-I); or gC2 mRNA, gD2 mRNA andgE2 mRNA given in combination (Trivalent-C). I indicates firstimmunization; II indicates second immunization.

FIG. 2C. gE2 Ab response as determined by antigen-specific ELISA in miceimmunized with gD2 mRNA; or gC2 mRNA, gD2 mRNA and gE2 mRNA each givenat a different intradermal site (Trivalent-I); or gC2 mRNA, gD2 mRNA andgE2 mRNA given in combination (Trivalent-C). I indicates firstimmunization; II indicates second immunization.

FIG. 3A. Antigen-specific IgG1 responses in mRNA vaccinated mice.Antibodies were evaluated after the first and second immunization forIgG1 responses. I indicates first immunization; II indicates secondimmunization.

FIG. 3B. Antigen-specific IgG2a responses in mRNA vaccinated mice.Antibodies were evaluated after the first and second immunization forIgG2a responses. I indicates first immunization; II indicates secondimmunization.

FIG. 4 . Neutralizing antibody titers in mRNA vaccinated mice. 50%endpoint neutralization titers of serum were obtained after the secondimmunization. Titers were performed using 10% human complement.Trivalent-I animals were immunized with gC2/liposomal nanoparticle(LNP), gD2/LNP, and gE2/LNP, each given at a different site. Trivalent-Canimals were immunized with gC2, gD2 and gE2 combined into a single LNP.P values comparing 50% endpoint neutralizing titers: Trivalent-I versusgD2, p=0.04; Trivalent-C versus gD2, p=0.002; Trivalent-I versusTrivalent-C, p-0.026.

FIGS. 5A-5B. CD4⁺ T cell responses to gC2, gD2 and gE2 mRNA eachadministered at a different intradermal site. Splenocytes werestimulated with subunit antigen glycoproteins (FIG. 5A) or 15 amino acidpeptides with 11 overlapping amino acids to stimulate HSV-2 specific Tcell responses (FIG. 5B). * indicates p<0.05 (t test) comparing gC, gDor gE with PBS stimulated CD4⁺ T cells or DMSO stimulated CD4⁺ T cells.Error bars represent SD.

FIGS. 6A-6B. CD8⁺ T cell responses to gC2, gD2 and gE2 mRNA, eachadministered at a different intradermal site. Splenocytes werestimulated with subunit antigen glycoproteins (FIG. 6A) or 15 amino acidpeptides with 11 overlapping amino acids to stimulate HSV-2 specific Tcell responses (FIG. 6B). * indicates p<0.05 comparing gE pool 2 withDMSO control. Error bars represent SD.

FIG. 7A. Survival in BALB/c mice immunized with mRNA twice at 28 dayintervals and challenged intravaginally with HSV-2. Trivalent-Irepresents animals immunized with gC2/LNP, gD2/LNP, and gE2/LNP eachgiven at different intradermal sites. Trivalent-C represents animalsimmunized with gC2, gD2 and gE2 combined into a single LNP forimmunization.

FIG. 7B. Weight loss (−) or gain (+) and neurological signs in BALB/cmice immunized with mRNA twice at 28 day intervals and challengedintravaginally with HSV-2. Trivalent-I represents animals immunized withgC2/LNP, gD2/LNP, and gE2/LNP each given at different intradermal sites.Trivalent-C represents animals immunized with gC2, gD2 and gE2 combinedinto a single LNP for immunization.

FIGS. 8A-8B. Vaginal viral titers in mRNA vaccinated mice afterintravaginal challenge with HSV-2. Vaginal swab titers were obtained onDay 2 (FIG. 8A) and Day 4 (FIG. 8B) post-challenge. The dotted linesindicate the limit of detection of the assay at 7 PFU/ml.

FIG. 9 . Genital disease in mRNA-vaccinated mice after HSV-2 vaginalchallenge. Mice were immunized with poly C as a control, or with gD2mRNA/LNP, trivalent at individual sites for each glycoprotein mRNA(Trivalent-I) or trivalent with all three mRNAs combined (Trivalent-C).Genital disease was scored for 28 days. All animals in the poly C groupdied by day 10. ***, indicates p<0.001 comparing poly C with the other 3groups.

FIG. 10 . HSV-2 DNA copies in dorsal root ganglia (DRG) in mRNAvaccinated mice on day 4 post challenge. HSV-2 DNA in DRG was measuredby qPCR. DRG from 4 to 5 animals per group were evaluated for HSV-2 DNAat 4 days post challenge. The bars represent the mean values per group.

FIG. 11A. The trivalent mRNA-LNP vaccine induces a potent T follicularhelper cell response in mice. BALB/c female mice were left un-immunizedas naïve control animals or immunized intradermally twice at 28 dayintervals with poly C mRNA-LNP or trivalent modified mRNA-LNP. The polyC mRNA controls received 10 μg Poly C mRNA-LNP divided into 4 aliquotsand administered at 4 separate sites. The trivalent modified mRNA groupreceived 10 μg gC2 mRNA-LNP, 10 μg gD2 mRNA-LNP, and 10 μg gE2 mRNA-LNPeach divided into 2 aliquots and each given at 2 sites. Two weeks afterthe second immunization, spleens were harvested from 5 animals per groupand flow cytometry performed to detect T follicular helper (Tfh) cellresponses (*p<0.05).

FIG. 11B. The trivalent mRNA-LNP vaccine induces a potent germinalcenter B cell response in mice. BALB/c female mice were leftun-immunized as naïve control animals or immunized intradermally twiceat 28 day intervals with poly C mRNA-LNP or trivalent modified mRNA-LNP.The poly C mRNA controls received 10 μg Poly C mRNA-LNP divided into 4aliquots and administered at 4 separate sites. The trivalent modifiedmRNA group received 10 μg gC2 mRNA-LNP, 10 μg gD2 mRNA-LNP, and 10 μggE2 mRNA-LNP each divided into 2 aliquots and each given at 2 sites. Twoweeks after the second immunization, spleens were harvested from 5animals per group and flow cytometry performed to detect germinal centerB cell responses (*p<0.05).

FIGS. 12A-12C. Genital mucosa IgG antibody responses. BALB/c mice wereimmunized intradermally twice at 28 day intervals with 10 μg of ploy CmRNA-LNP, 10 μg gD2 mRNA-LNP or 10 μg each of gC2, gD2, gE trivalentmodified mRNA-LNP. The trivalent mRNA was combined and administered as10 μg gC2 mRNA & 10 μg gD2 mRNA & 10 μg gE2 mRNA combined into LNP anddivided into 4 aliquots and given at 4 sites. One month after the secondimmunization, 60 μl of media was introduced in the vaginal cavity andretrieved. IgG titers were determined at a 1:50 dilution of the vaginalwash fluids by ELISA to gC2 (FIG. 12A), gD2 (FIG. 12B), and gE2 (FIG.12C) (n=10 mice in the poly C group, n=10 in the gD2 mRNA group and n=25in the trivalent mRNA group; ***p<0.001; **p<0.01).

FIG. 13 . The trivalent mRNA-LNP vaccine produces antibodies that blockgC2 binding to complement component C3b. BALB/c mice were leftunimmunized as a source of non-immune IgG, or immunized intradermallywith poly C mRNA-LNP or trivalent mRNA-LNP. The poly C mRNA controlsreceived 10 μg poly C mRNA-LNP divided into 4 aliquots and administeredat 4 separate sites. The gD2 mRNA group received 10 μg gD2 mRNA-LNPadministered as described for the poly C mRNA-LNP. The trivalentmodified mRNA group received 10 μg gC2 mRNA-LNP, 10 μg gD2 mRNA-LNP, and10 μg gE2 mRNA-LNP combined into LNP and divided into 4 aliquots andgiven at 4 sites. There were 10 mice in each group. Sera from the 10mice were pooled and IgG was purified. The IgG was evaluated at 12μg/200 μl for its ability to block complement component C3b binding togC2. (****p<0.0001).

FIGS. 14A-14F. The trivalent mRNA vaccine provides outstandingprotection in mice when the vaccine is administered intramuscularly.BALB/c mice were immunized intramuscularly with poly C mRNA-LNP as acontrol (15/group) or with trivalent mRNA containing 10 μg each of gC2,gD2 and gE2 mRNA-LNP (20/group). FIG. 14A presents data on mousesurvival; FIG. 14B presents data on weight loss; FIG. 14C presents dataon genital disease. DRG were harvested from nine poly C animals at thetime of euthanasia between days 7 and 12 post-infection or at the end ofthe experiment on day 28 in the trivalent mRNA group. FIG. 14D presentsdata on HSV-2 DNA in DRG. FIG. 14E presents data on vaginal viralcultures on Day 2 and FIG. 14F presents data on vaginal viral cultureson Day 4. Difference between poly C and trivalent groups aresignificant, p<0.001 for FIGS. 14A-14F.

FIGS. 15A-15C. The trivalent mRNA vaccine is highly efficacious in theguinea pig genital infection model. Hartley Strain female guinea pigswere left unimmunized and uninfected (naive group, n=10), immunizedthree times intradermally at monthly intervals with 20 μg poly CmRNA-LNP (n=10) or with 20 μg each of gC2, gD2, gE modified mRNA-LNP(n=10). One month after the final immunization, animals in the poly Cand trivalent mRNA groups were infected intravaginally with 5×10⁵ PFU ofHSV-2 strain MS (50 LD₅₀). Animals were observed for death, genitallesions during the acute phase of infection (days 1-14) and genitallesions during the recurrent phase of infection (days 15-60). FIG. 15Apresents data on survival; FIG. 15B provides data on vaginal disease(acute phase); and FIG. 15C provides data on vaginal disease (recurrentphase).

DETAILED DESCRIPTION OF THE INVENTION

Compositions

In one embodiment, the present invention provides compositionscomprising one or more modified mRNAs, wherein each of said modifiedmRNAs encodes a Herpes Simplex Virus (HSV) glycoprotein or immunogenicfragment thereof.

In one embodiment, the present invention provides a compositioncomprising one or more nucleoside modified mRNAs, wherein each of saidmodified mRNAs encodes a Herpes Simplex Virus (HSV) glycoprotein orimmunogenic fragment thereof, and wherein said modified mRNA comprisesone or more pseudouridine or pseudouridine family residues.

In one embodiment, the HSV glycoprotein comprises glycoprotein D (gD),glycoprotein C (gC), glycoprotein E (gE), glycoprotein B (gB),glycoprotein H (gH), glycoprotein L (gL) glycoprotein I (gI), or acombination thereof.

Thus, in one embodiment, the present invention provides a compositioncomprising one or more modified mRNAs encoding HSV gD, gC, gE, gB, gH,gL, gI, or immunogenic fragments thereof. In one embodiment, themodified mRNAs comprise pseudouridine-modified mRNAs.

In one embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gD or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gC or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gE or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gB or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gH or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gL or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV gI or fragment thereof.

In one embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV gD or fragment thereof; and(b) a modified mRNA encoding HSV gC or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV gD or fragment thereof; and(b) a modified mRNA encoding HSV gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV gC or fragment thereof; and(b) a modified mRNA encoding HSV gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV gD or fragment thereof; (b)a modified mRNA encoding HSV gC or fragment thereof, and (c) a modifiedmRNA encoding HSV gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV gD or fragment thereof; (b)a modified mRNA encoding HSV gC or fragment thereof, (c) a modified mRNAencoding HSV gE or fragment thereof; and (d) a modified mRNA encodingHSV gB or fragment thereof.

In one embodiment, the HSV glycoproteins are HSV-2 glycoproteins. Inanother embodiment, the HSV glycoproteins are HSV-1 glycoproteins. Inone embodiment, the HSV glycoproteins comprise both HSV-2 glycoproteinsand HSV-1 glycoproteins. In another embodiment, the HSV glycoproteinscomprise a mixture of HSV-2 glycoproteins and HSV-1 glycoproteins.

In one embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gD or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gC or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gE or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gE or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gB or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gH or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gL or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-2 gI or fragment thereof.

In one embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-2 gD or fragment thereof;and (b) a modified mRNA encoding HSV-2 gC or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-2 gD or fragment thereof;and (b) a modified mRNA encoding HSV-2 gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-2 gC or fragment thereof;and (b) a modified mRNA encoding HSV-2 gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-2 gD or fragment thereof;(b) a modified mRNA encoding HSV-2 gC or fragment thereof, and (c) amodified mRNA encoding HSV-2 gE or fragment thereof.

In one embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gD or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gC or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gE or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gE or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gB or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gH or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gL or fragment thereof. Inanother embodiment, the present invention provides compositionscomprising a modified mRNA encoding HSV-1 gI or fragment thereof.

In one embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-1 gD or fragment thereof;and (b) a modified mRNA encoding HSV-1 gC or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-1 gD or fragment thereof;and (b) a modified mRNA encoding HSV-1 gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-1 gC or fragment thereof;and (b) a modified mRNA encoding HSV-1 gE or fragment thereof.

In another embodiment, the present invention provides a compositioncomprising: (a) a modified mRNA encoding HSV-1 gD or fragment thereof;(b) a modified mRNA encoding HSV-1 gC or fragment thereof, and (c) amodified mRNA encoding HSV-1 gE or fragment thereof.

In one embodiment, any of the compositions as described herein consistsessentially of one or more modified mRNAs, wherein each of said modifiedmRNAs encodes an HSV glycoprotein or immunogenic fragment thereof. Inanother embodiment, any of the compositions as described herein consistsof one or more modified mRNAs, wherein each of said modified mRNAsencodes an HSV glycoprotein or immunogenic fragment thereof.

In another embodiment, the present invention provides compositionscomprising a modified mRNA encoding an HSV gD protein, a modified mRNAencoding an HSV gC protein, a modified mRNA encoding an HSV gE proteinand modified mRNAs encoding one or more additional HSV glycoproteins. Inone embodiment, said additional HSV glycoproteins comprise gB orimmunogenic fragment thereof, gH or immunogenic fragment thereof, gL orimmunogenic fragment thereof, gI or immunogenic fragment thereof, or anycombination thereof. In one embodiment, said additional HSVglycoproteins comprise glycoprotein M (gM), glycoprotein N (gN),glycoprotein K (gK), glycoprotein G (gG), glycoprotein J (gJ), or animmunogenic fragment thereof.

In one embodiment, compositions of the present invention and for use inthe methods of the present invention comprise both HSV-2 glycoproteinsor glycoprotein fragments and HSV-1 glycoproteins or glycoproteinfragments. In another embodiment, compositions of the present inventionand for use in the methods of the present invention comprise a mixtureof HSV-2 glycoproteins or glycoprotein fragments and HSV-1 glycoproteinsor glycoprotein fragments. For example, in one embodiment, a compositionof the present invention comprises HSV-2 gC, HSV-1 gD, and HSV-2 gE, orfragments thereof. In another embodiment, a composition of the presentinvention comprises HSV-1 gC, HSV-2 gD, and HSV-2 gE, or fragmentsthereof. In another embodiment, a composition of the present inventioncomprises HSV-2 gC, HSV-2 gD, and HSV-1 gE, or fragments thereof. Inanother embodiment, a composition of the present invention comprisesHSV-1 gC, HSV-1 gD, and HSV-2 gE, or fragments thereof. In anotherembodiment, a composition of the present invention comprises HSV-1 gC,HSV-2 gD, and HSV-1 gE, or fragments thereof. In another embodiment, acomposition of the present invention comprises HSV-2 gC, HSV-1 gD, andHSV-1 gE, or fragments thereof.

In another embodiment, the compositions of the present inventioncomprise one or more additional HSV-1 glycoproteins or HSV-2glycoproteins or both HSV-1 and HSV-2 glycoproteins, as describedherein. For example, in one embodiment, a composition of the presentinvention comprising HSV-2 gC, HSV-1 gD, and HSV-2 gE may furthercomprise HSV-1 gI. In another embodiment, a composition of the presentinvention comprising HSV-2 gC, HSV-2 gD, and HSV-2 gE may furthercomprise HSV-1 gB. Each of the possible combinations of HSV-1 and HSV-2glycoproteins represents a separate embodiment of the invention.

“Encoding” refers, in one embodiment, to an RNA molecule that contains agene that encodes the protein of interest. In another embodiment, theRNA molecule comprises a protein coding sequence that encodes theprotein of interest. In another embodiment, one or more other proteinsis also encoded. In another embodiment, the protein of interest is theonly protein encoded. Each possibility represents a separate embodimentof the present invention.

“Immunogenic fragment” refers, in another embodiment, to a portion of aprotein that is immunogenic and elicits a protective immune responsewhen administered to a subject.

In one embodiment, “immunogenicity” or “immunogenic” is used herein torefer to the innate ability of a protein, peptide, nucleic acid, antigenor organism to elicit an immune response in an animal when the protein,peptide, nucleic acid, antigen or organism is administered to theanimal. Thus, “enhancing the immunogenicity” in one embodiment, refersto increasing the ability of a protein, peptide, nucleic acid, antigenor organism to elicit an immune response in an animal when the protein,peptide, nucleic acid, antigen or organism is administered to an animal.The increased ability of a protein, peptide, nucleic acid, antigen ororganism to elicit an immune response can be measured by, in oneembodiment, a greater number of antibodies to a protein, peptide,nucleic acid, antigen or organism, a greater diversity of antibodies toan antigen or organism, a greater number of T-cells specific for aprotein, peptide, nucleic acid, antigen or organism, a greater cytotoxicor helper T-cell response to a protein, peptide, nucleic acid, antigenor organism, and the like.

In one embodiment, an immunogenic polypeptide is also antigenic.“Antigenic” refers, in another embodiment, to a peptide capable ofspecifically interacting with an antigen recognition molecule of theimmune system, e.g. an immunoglobulin (antibody) or T cell antigenreceptor. An antigenic peptide contains, in another embodiment, anepitope of at least about 8 amino acids (AA). An antigenic portion of apolypeptide, also called herein the epitope in one embodiment, can bethat portion that is immunodominant for antibody or T cell receptorrecognition, or it can be a portion used to generate an antibody to themolecule by conjugating the antigenic portion to a carrier polypeptidefor immunization. A molecule that is antigenic need not be itselfimmunogenic, i.e., capable of eliciting an immune response without acarrier.

In one embodiment, “functional” within the meaning of the invention, isused herein to refer to the innate ability of a protein, peptide,nucleic acid, fragment or a variant thereof to exhibit a biologicalactivity or function. In one embodiment, such a biological function isits binding property to an interaction partner, e.g., amembrane-associated receptor, and in another embodiment, itstrimerization property. In the case of functional fragments and thefunctional variants of the invention, these biological functions may infact be changed, e.g., with respect to their specificity or selectivity,but with retention of the basic biological function.

In one embodiment, the term “fragment” is used herein to refer to aprotein or polypeptide that is shorter or comprises fewer amino acidsthan the full length protein or polypeptide. In another embodiment,fragment refers to a nucleic acid encoding the protein fragment that isshorter or comprises fewer nucleotides than the full length nucleicacid. In another embodiment, the fragment is an N-terminal fragment. Inanother embodiment, the fragment is a C-terminal fragment. In oneembodiment, the fragment is an intrasequential section of the protein,peptide, or nucleic acid. In another embodiment, the fragment is animmunogenic intrasequential section of the protein, peptide or nucleicacid. In another embodiment, the fragment is a functionalintrasequential section within the protein, peptide or nucleic acid. Inanother embodiment, the fragment is an N-terminal immunogenic fragment.In one embodiment, the fragment is a C-terminal immunogenic fragment. Inanother embodiment, the fragment is an N-terminal functional fragment.In another embodiment, the fragment is a C-terminal functional fragment.In another embodiment, the fragment contains pieces of the proteinlinked together or pieces of multiple proteins linked together.

Thus, in one embodiment, an “immunogenic fragment” of a protein asdescribed in the present invention refers to a portion of the proteinthat is immunogenic, in one embodiment and in another embodiment,elicits a protective immune response when administered to a subject.

In another aspect, the present invention provides compositionscomprising modified mRNAs, wherein each of said modified mRNAs encodesa) HSV glycoprotein D (gD) or an immunogenic fragment thereof, b) HSVglycoprotein C (gC) or an immunogenic fragment thereof, c) HSVglycoprotein E (gE) or an immunogenic fragment thereof, or anycombination thereof.

In one embodiment, the present invention provides a compositioncomprising a modified mRNA encoding an HSV gD or an immunogenic fragmentthereof, a modified mRNA encoding an HSV gC or an immunogenic fragmentthereof, and a modified mRNA encoding an HSV gE or an immunogenicfragment thereof.

In one embodiment, compositions of modified mRNA encoding gD-1 areprotective against HSV-1 infection. Further, combination compositions ofmodified mRNA encoding gC-1/gD-1/gE-1 confer superior protectioncompared with compositions containing modified mRNA encoding gC-1 alone,gD-1 alone, or gE-1 alone. Further, as provided herein, compositions ofmodified mRNA encoding gD-2 are protective against HSV-2 infection(FIGS. 7-10 ). Further, combination compositions of modified mRNAencoding gC-2/gD-2/gE-2 confer superior protection compared withcompositions containing modified mRNA encoding gC-2 alone, gD-2 alone,or gE-2 alone.

In another embodiment, inclusion of a modified mRNA encoding gC, and/ora modified mRNA encoding gE in the composition of the present inventionincreases the efficaciousness of anti-gD antibodies elicited by thecomposition. In another embodiment, inclusion of a modified mRNAencoding gC, and/or a modified mRNA encoding gE in the composition ofthe present invention increases the dose of modified mRNA encoding gDrequired to elicit antibodies that inhibit binding of gD to a cellularreceptor. In another embodiment, inclusion of a modified mRNA encodinggC, and/or a modified mRNA encoding gE in the composition of the presentinvention decreases the dose of modified mRNA encoding gD required toelicit antibodies that inhibit binding of gD to a cellular receptor whena dose of modified mRNA encoding a gD is administered separately frommodified mRNAs encoding the gC protein or gE protein.

In another embodiment, inclusion of a modified mRNA encoding gC, and/ora modified mRNA encoding gE in the composition of the present inventionenhances the effectiveness of an innate immune response. In anotherembodiment, the innate immune response is an antibody-mediated immuneresponse. In another embodiment, the innate immune response is anon-antibody-mediated immune response. In another embodiment, the innateimmune response is an NK (natural killer) cell response. In anotherembodiment, the innate immune response is any other innate immuneresponse known in the art.

In another embodiment, inclusion of a modified mRNA encoding gC, and/ora modified mRNA encoding gE in the composition of the present inventionincreases the efficaciousness of antibodies elicited by the compositionagainst one of the above glycoproteins. In another embodiment, inclusionof a modified mRNA encoding gC, and/or a modified mRNA encoding gE inthe composition of the present invention decreases the dose of one ofthe above glycoproteins required to elicit antibodies that inhibitbinding of the glycoprotein to a cellular receptor thereof, when a doseof one of the glycoproteins is administered separately from one of theother glycoproteins.

Glycoprotein D

In one embodiment, a composition of the present invention comprises amodified mRNA encoding HSV-1 gD protein. In another embodiment, thecomposition comprises a modified mRNA encoding a fragment of an HSV-1 gDprotein.

In one embodiment, the nucleotide sequence of the modified mRNA encodingan HSV-1 gD fragment comprises:

(SEQ ID NO: 1) GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGC AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACUCCAAGUACGCCCUGGCCGACGCCUCCCUGAAGAUGGCCGACCCCAACCGCUUCCGCGGCAAGGACCUGCCCGUGCUGGACCAGCUGACCGACCCCCCCGGCGUGCGCCGCGUGUACCACAUCCAGGCCGGCCUGCCCGACCCCUUCCAGCCCCCCUCCCUGCCCAUCACCGUGUACUACGCCGUGCUGGAGCGCGCCUGCCGCUCCGUGCUGCUGAACGCCCCCUCCGAGGCCCCCCAGAUCGUGCGCGGCGCCUCCGAGGACGUGCGCAAGCAGCCCUACAACCUGACCAUCGCCUGGUUCCGCAUGGGCGGCAACUGCGCCAUCCCCAUCACCGUGAUGGAGUACACCGAGUGCUCCUACAACAAGUCCCUGGGCGCCUGCCCCAUCCGCACCCAGCCCCGCUGGAACUACUACGACUCCUUCUCCGCCGUGUCCGAGGACAACCUGGGCUUCCUGAUGCACGCCCCCGCCUUCGAGACCGCCGGCACCUACCUGCGCCUGGUGAAGAUCAACGACUGGACCGAGAUCACCCAGUUCAUCCUGGAGCACCGCGCCAAGGGCUCCUGCAAGUACGCCCUGCCCCUGCGCAUCCCCCCCUCCGCCUGCCUGUCCCCCCAGGCCUACCAGCAGGGCGUGACCGUGGACUCCAUCGGCAUGCUGCCCCGCUUCAUCCCCGAGAACCAGCGCACCGUGGCCGUGUACUCCCUGAAGAUCGCCGGCUGGCACGGCCCCAAGGCCCCCUACACCUCCACCCUGCUGCCCCCCGAGCUGUCCGAGACCCCCAACGCCACCCAGCCCGAGCUGGCCCCCGAGGACCCCGAGGACUCCGCCCUGCUGGAGGACCCCGUGGGCACCGUGGCCCCCCAGAUCCCCCCCAACUGGCACAUCCCCUCCAUCCAGGACGCCGCCACCCCCUACUAACUAGUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC

In one embodiment, all uridine residues are 1-methyl-pseudouridine. Inone embodiment, underlined residues represent 5′ untranslated sequences.In one embodiment, bold residues represent a signal sequence (leadersequence) to assist expression of the gD1 fragment. In one embodiment,italicized residues represent 3′ untranslated sequences and polyadenylation tail.

In another embodiment, the nucleotide sequence of the modified mRNAencoding an HSV-1 gD fragment lacks the 5′ untranslated sequences, thesignal sequence, the 3′ untranslated sequences, the poly adenylationtail, or a combination thereof.

In one embodiment, the HSV-1 gD fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises amino acids 26-331 of gD from HSV-1 Patton strain, as setforth in the following amino acid sequence:

(SEQ ID NO: 2) KYALADASLKMADPNRFRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQ DAATPY 

In one embodiment, the full length HSV-1 gD encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises the following amino acid sequence:

(SEQ ID NO: 3) MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKLADPNRFRRKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPKAPYTSTLLPPELSETPNATQPELAPEAPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMRRRTQKAPKRIRLPHIREDDQPSSHQPLFY

In another embodiment, the HSV-1 gD encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in any one of the following GenBankAccession Numbers: AAL90884.1 (KHS2 strain), AAL90883.1 (KHS1 strain),AAK93950.1 (F strain), AAB59754.1 (F strain), AAA19631.1 (mutant strainnot identified), AAA19630.1 (mutant strain not identified), orAAA19629.1 (strain not identified).

In another embodiment, the HSV-1 gD encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in any of the following GenBankAccession Numbers: A1Z0Q5.2, AAA45780.1, AAA45785.1, AAA45786.1,AAA96682.1, AAK19597.1, AAN74642.1, ABI63524.1, ABM52978.1, ABM52979.1,ABM52980.1, ABM52981.1, ABM66847.1, ABM66848.1, ACM62295.1, ADD60053.1,ADD60130.1, ADM22389.1, ADM22466.1, ADM22542.1, ADM22619.1, ADM22696.1,ADM22773.1, ADM22849.1, ADM22926.1, ADM23003.1, ADM23079.1, ADM23155.1,ADM23231.1, ADM23309.1, ADM23383.1, ADM23457.1, ADM23531.1, ADM23605.1,ADM23680.1, ADM23755.1, ADM23831.1, AEQ77097.1, AER37647.1, AER37715.1,AER37786.1, AER37857.1, AER37929.1, AER38000.1, AER38070.1, AFE62894.1,AFH41180.1, AFI23657.1, AFK50415.1, AFP86430.1, AGZ01928.1, AIR95858.1,AJE60009.1, AJE60080.1, AJE60151.1, AJE60222.1, AJE60293.1, AJE60439.1,AKE48645.1, AKG59246.1, AKG59318.1, AKG59391.1, AKG59462.1, AKG59536.1,AKG59609.1, AKG59682.1, AKG59755.1, AKG59826.1, AKG59898.1, AKG59972.1,AKG60046.1, AKG60118.1, AKG60189.1, AKG60261.1, AKG60334.1, AKG60404.1,AKG60474.1, AKG60546.1, AKG60620.1, AKG60692.1, AKG60763.1, AKG60835.1,AKG60906.1, AKG60978.1, AKG61050.1, AKG61123.1, AKG61194.1, AKG61267.1,AKG61339.1, AKG61411.1, AKG61484.1, AKG61556.1, AKG61629.1, AKG61703.1,AKG61774.1, AKG61847.1, AKG61920.1, AKG61993.1, AKH80463.1, AKH80536.1,ALM22635.1, ALM22709.1, ALM22783.1, ALM22857.1, ALO18662.1, ALO18738.1,AMB65662.1, AMB65735.1, AMB65809.1, AMB65885.1, AMB65956.1, AMN09832.1,ANN83964.1, ANN84041.1, ANN84117.1, ANN84194.1, ANN84271.1, ANN84348.1,ANN84424.1, ANN84500.1, ANN84577.1, ANN84653.1, ANN84730.1, ANN84806.1,ANN84883.1, ANN84959.1, ANN85036.1, ANN85112.1, ANN85187.1, ANN85264.1,ANN85341.1, ANN85416.1, ANN85494.1, ANN85571.1, ANN85648.1, ANN85724.1,ANN85801.1, AOY34093.1, AOY34141.1, AOY34243.1, AOY34271.1, AOY34337.1,AOY36685.1, ARB08957.1, ARO37961.1, ARO37962.1, ARO37963.1, ARO37964.1,ARO37965.1, ARO37966.1, ARO37967.1, ARO37968.1, ARO37969.1, ARO37970.1,ARO37971.1, ARO37972.1, ARO37973.1, ARO37974.1, ARO37975.1, ARO37976.1,ARO37977.1, ARO37978.1, ARO37979.1, ARO37980.1, ARO37981.1, ARO37982.1,ARO37983.1, ARO37984.1, ARO37985.1, ARO37986.1, ARO37987.1, ARO37988.1,ARO37989.1, ARO37990.1, ARO37991.1, ARO37992.1, ARO37993.1, ARO37994.1,ARO37995.1, ARO37996.1, ARO37997.1, ARO37998.1, ARO37999.1, ASM47664.1,ASM47741.1, ASM47818.1, ASM47893.1, BAM73419.1, CAA26060.1, CAA32283.1,CAA32284.1, CAA32289.1, CAA38245.1, CAT05431.1, P06476.1, P36318.1,P57083.1, P68331.1, Q05059.1, Q69091.1, SBO07792.1, SBO07819.1,SBO07855.1, SBO07869.1, SBO07887.1, SBO07908.1, SBS69553.1, SBS69561.1,SBS69579.1, SBS69625.1, SBS69688.1, SBS69694.1, SBS69717.1, SBS69727.1,SBS69811.1, SBT69395.1, SCL76902.1, VGBEDZ, or YP_009137141.1.

In another embodiment, the composition comprises a modified mRNAencoding an HSV-2 gD protein. In another embodiment, the compositioncomprises a modified mRNA encoding a fragment of an HSV-2 gD protein.

In one embodiment, the nucleotide sequence of the modified mRNA encodingan HSV-2 gD fragment comprises:

(SEQ ID NO: 4) GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGC AUGACCCGCCUGACCGUGCUGGCCCUGCUGGCCGGCCUGCUGGCCUCCUCCCGCGCCAAGUACGCCCUGGCCGACCCCUCCCUGAAGAUGGCCGACCCCAACCGCUUCCGCGGCAAGAACCUGCCCGUGCUGGACCAGCUGACCGACCCCCCCGGCGUGAAGCGCGUGUACCACAUCCAGCCCUCCCUGGAGGACCCCUUCCAGCCCCCCUCCAUCCCCAUCACCGUGUACUACGCCGUGCUGGAGCGCGCCUGCCGCUCCGUGCUGCUGCACGCCCCCUCCGAGGCCCCCCAGAUCGUGCGCGGCGCCUCCGACGAGGCCCGCAAGCACACCUACAACCUGACCAUCGCCUGGUACCGCAUGGGCGACAACUGCGCCAUCCCCAUCACCGUGAUGGAGUACACCGAGUGCCCCUACAACAAGUCCCUGGGCGUGUGCCCCAUCCGCACCCAGCCCCGCUGGUCCUACUACGACUCCUUCUCCGCCGUGUCCGAGGACAACCUGGGCUUCCUGAUGCACGCCCCCGCCUUCGAGACCGCCGGCACCUACCUGCGCCUGGUGAAGAUCAACGACUGGACCGAGAUCACCCAGUUCAUCCUGGAGCACCGCGCCCGCGCCUCCUGCAAGUACGCCCUGCCCCUGCGCAUCCCCCCCGCCGCCUGCCUGACCUCCAAGGCCUACCAGCAGGGCGUGACCGUGGACUCCAUCGGCAUGCUGCCCCGCUUCAUCCCCGAGAACCAGCGCACCGUGGCCCUGUACUCCCUGAAGAUCGCCGGCUGGCACGGCCCCAAGCCCCCCUACACCUCCACCCUGCUGCCCCCCGAGCUGUCCGACACCACCAACGCCACCCAGCCCGAGCUGGUGCCCGAGGACCCCGAGGACUCCGCCCUGCUGGAGGACCCCGCCGGCACCGUGUCCUCCCAGAUCCCCCCCAACUGGCACAUCCCCUCCAUCCAGGACGUGGCCCCCCACCACUAACUAGUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC 

In one embodiment, all uridine residues are 1-methyl-pseudouridine. Inone embodiment, underlined residues represent 5′ untranslated sequences.In one embodiment, bold residues represent a signal sequence (leadersequence) to assist expression of the gD2 fragment. In one embodiment,italicized residues represent 3′ untranslated sequences and polyadenylation tail.

In another embodiment, the nucleotide sequence of the modified mRNAencoding an HSV-2 gD fragment lacks the 5′ untranslated sequences, thesignal sequence, the 3′ untranslated sequences, the poly adenylationtail, or a combination thereof.

In one embodiment, the HSV-2 gD fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises amino acids 26-331 of gD from HSV-2 strain 333, as set forthin the following amino acid sequence:

(SEQ ID NO: 5) KYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQ DVAPHH.

In one embodiment, the full length HSV-2 gD encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises the following amino acid sequence:

(SEQ ID NO: 6) MGRLTSGVGTAALLVVAVGLRVVCAKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPGLIIGALAGSTLAVLVIGGIAFWVRRRAQMAPKRLRLPHIRDDDAPPSHQPLFY.

In another embodiment, the HSV-2 gD encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in GenBank Accession Numbers:1003204A, AAA45841.1, AAA45842.1, AAB60552.1, AAB60553.1, AAB60554.1,AAB60555.1, AAB72102.1, AAS01730.1, AAW23130.1, AAW23131.1, AAW23132.1,AAW23133.1, AAW23134.1, ABS84899.1, ABU45433.1, ABU45434.1, ABU45435.1,ABU45461.1, ABU45462.1, ACA28831.1, AEV91405.1, AFM93876.1, AFS18198.1,AFS18199.1, AFS18200.1, AFS18201.1, AFS18202.1, AFS18203.1, AFS18204.1,AFS18205.1, AFS18206.1, AFS18207.1, AFS18208.1, AFS18209.1, AFS18210.1,AFS18211.1, AFS18212.1, AFS18213.1, AFS18214.1, AFS18215.1, AFS18216.1,AFS18217.1, AFS18218.1, AFS18219.1, AFS18220.1, AFS18221.1, AHG54730.1,AIL27720.1, AIL27721.1, AIL27722.1, AIL27723.1, AIL27724.1, AIL27725.1,AIL27726.1, AIL27727.1, AIL27728.1, AIL27729.1, AIL27730.1, AIL27731.1,AIL28069.1, AIL28070.1, AKC42828.1, AKC59305.1, AKC59376.1, AKC59447.1,AKC59518.1, AKC59589.1, AMB66102.1, AMB66171.1, AMB66244.1, AMB66321.1,AMB66394.1, AMB66463.1, AQZ55754.1, AQZ55825.1, AQZ55896.1, AQZ55967.1,AQZ56038.1, AQZ56109.1, AQZ56180.1, AQZ56251.1, AQZ56322.1, AQZ56393.1,AQZ56464.1, AQZ56535.1, AQZ56606.1, AQZ56677.1, AQZ56748.1, AQZ56819.1,AQZ56890.1, AQZ56961.1, AQZ57032.1, AQZ57103.1, AQZ57174.1, AQZ57245.1,AQZ57316.1, AQZ57387.1, AQZ57458.1, AQZ57529.1, AQZ57600.1, AQZ57671.1,AQZ57742.1, AQZ57813.1, AQZ57884.1, AQZ57955.1, AQZ58026.1, AQZ58097.1,AQZ58168.1, AQZ58239.1, AQZ58310.1, AQZ58381.1, AQZ58452.1, AQZ58523.1,AQZ58594.1, AQZ58665.1, AQZ58736.1, AQZ58807.1, AQZ58878.1, AQZ58949.1,AQZ59020.1, AQZ59091.1, AQZ59162.1, ARO38000.1, ARO38001.1, ARO38002.1,ARO38003.1, ARO38004.1, ARO38005.1, ARO38006.1, ARO38007.1, ARO38008.1,ARO38009.1, ARO38010.1, ARO38011.1, ARO38012.1, ARO38013.1, ARO38014.1,ARO38015.1, ARO38016.1, ARO38017.1, ARO38018.1, ARO38019.1, ARO38020.1,ARO38021.1, ARO38022.1, ARO38023.1, ARO38024.1, ARO38025.1, ARO38026.1,ARO38027.1, ARO38028.1, ARO38029.1, ARO38030.1, ARO38031.1, ARO38032.1,ARO38033.1, ARO38034.1, ARO38035.1, ARO38036.1, ARO38037.1, ARO38038.1,ARO38039.1, ARO38040.1, ARO38041.1, ARO38042.1, ARO38043.1, ARO38044.1,CAA26025.1, CAB06713.1, CAC33573.1, CAT05432.1, P03172.2, Q69467.1, orYP_009137218.1.

In another embodiment, the gD protein or fragment includes Y63. Inanother embodiment, the gD protein or fragment includes R159. In anotherembodiment, the gD protein or fragment includes D240. In anotherembodiment, the gD protein or fragment includes P246. In anotherembodiment, the gD protein or fragment includes a residue selected fromY63, R159, D240, and P246. In another embodiment, inclusion of one ofthese residues elicits antibodies that inhibit binding to nectin-1.

The nomenclature used herein for gD amino acid residues includes theresidues of the signal sequence. Thus, residue one of the mature proteinis referred to as “26.”

Each modified mRNA encoding gD-1 and gD-2 protein or fragment thereofrepresents a separate embodiment of the present invention.

In another embodiment, the HSV gD, gC, and gE proteins, and fragmentsthereof, encoded by the modified mRNA as disclosed herein are describedin US Patent Publication No. 2013-0028925-A1, which is incorporated byreference herein in its entirety.

In another embodiment, a gD protein fragment encoded by modified mRNAutilized in the methods and compositions of the present invention is animmunogenic fragment. In another embodiment, a gD immunoprotectiveantigen need not be the entire protein. The protective immune responsegenerally involves, in another embodiment, an antibody response. Inanother embodiment, mutants, sequence conservative variants, andfunctional conservative variants of gD are useful in methods andcompositions of the present invention, provided that all such variantsretain the required immuno-protective effect. In another embodiment, theimmunogenic fragment can comprise an immuno-protective gD antigen fromany strain of HSV. In another embodiment, the immunogenic fragment cancomprise sequence variants of HSV, as found in infected individuals.

Glycoprotein C

In another embodiment, a composition of the present invention comprisesa modified mRNA encoding HSV-1 gC protein. In another embodiment, thecomposition comprises a modified mRNA encoding a fragment of an HSV-1 gCprotein.

In one embodiment, the nucleotide sequence of the modified mRNA encodingan HSV-1 gC fragment comprises:

(SEQ ID NO: 7) GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGC AUGGCCAUCUCCGGCGUGCCCGUGCUGGGCUUCUUCAUCAUCGCCGUGCUGAUGUCCGCCCAGGAGUCCUGGGCCGAGACCGCCUCCACCGGCCCCACCAUCACCGCCGGCGCCGUGACCAACGCCUCCGAGGCCCCCACCUCCGGCUCCCCCGGCUCCGCCGCCUCCCCCGAGGUGACCCCCACCUCCACCCCCAACCCCAACAACGUGACCCAGAACAAGACCACCCCCACCGAGCCCGCCUCCCCCCCCACCACCCCCAAGCCCACCUCCACCCCCAAGUCCCCCCCCACCUCCACCCCCGACCCCAAGCCCAAGAACAACACCACCCCCGCCAAGUCCGGCCGCCCCACCAAGCCCCCCGGCCCCGUGUGGUGCGACCGCCGCGACCCCCUGGCCCGCUACGGCUCCCGCGUGCAGAUCCGCUGCCGCUUCCGCAACUCCACCCGCAUGGAGUUCCGCCUGCAGAUCUGGCGCUACUCCAUGGGCCCCUCCCCCCCCAUCGCCCCCGCCCCCGACCUGGAGGAGGUGCUGACCAACAUCACCGCCCCCCCCGGCGGCCUGCUGGUGUACGACUCCGCCCCCAACCUGACCGACCCCCACGUGCUGUGGGCCGAGGGCGCCGGCCCCGGCGCCGACCCCCCCCUGUACUCCGUGACCGGCCCCCUGCCCACCCAGCGCCUGAUCAUCGGCGAGGUGACCCCCGCCACCCAGGGCAUGUACUACCUGGCCUGGGGCCGCAUGGACUCCCCCCACGAGUACGGCACCUGGGUGCGCGUGCGCAUGUUCCGCCCCCCCUCCCUGACCCUGCAGCCCCACGCCGUGAUGGAGGGCCAGCCCUUCAAGGCCACCUGCACCGCCGCCGCCUACUACCCCCGCAACCCCGUGGAGUUCGACUGGUUCGAGGACGACCGCCAGGUGUUCAACCCCGGCCAGAUCGACACCCAGACCCACGAGCACCCCGACGGCUUCACCACCGUGUCCACCGUGACCUCCGAGGCCGUGGGCGGCCAGGUGCCCCCCCGCACCUUCACCUGCCAGAUGACCUGGCACCGCGACUCCGUGACCUUCUCCCGCCGCAACGCCACCGGCCUGGCCCUGGUGCUGCCCCGCCCCACCAUCACCAUGGAGUUCGGCGUGCGCCACGUGGUGUGCACCGCCGGCUGCGUGCCCGAGGGCGUGACCUUCGCCUGGUUCCUGGGCGACGACCCCUCCCCCGCCGCCAAGUCCGCCGUGACCGCCCAGGAGUCCUGCGACCACCCCGGCCUGGCCACCGUGCGCUCCACCCUGCCCAUCUCCUACGACUACUCCGAGUACAUCUGCCGCCUGACCGGCUACCCCGCCGGCAUCCCCGUGCUGGAGCACCACUAACUAGUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC

In one embodiment, all uridine residues are 1-methyl-pseudouridine. Inone embodiment, underlined residues represent 5′ untranslated sequences.In one embodiment, bold residues represent a signal sequence (leadersequence) to assist expression of the gC1 fragment. In one embodiment,italicized residues represent 3′ untranslated sequences and polyadenylation tail.

In another embodiment, the nucleotide sequence of the modified mRNAencoding an HSV-1 gC fragment does not comprise the 5′ untranslatedsequences, the signal sequence, the 3′ untranslated sequences, the polyadenylation tail, or a combination thereof.

In one embodiment, the HSV-1 gC fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises amino acids 27-457 of gC from HSV-1 KOS strain, as set forthin the following amino acid sequence:

(SEQ ID NO: 8) ETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMYYLAWGRMDSPHEYGTWVRVRMFRPPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHH.

In one embodiment, the gC fragment encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises aminoacids 27-457 of gC from an HSV-1 strain.

In one embodiment, the full length HSV-1 gC encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises the following amino acid sequence:

(SEQ ID NO: 9) MAPGRVGLAVVLWGLLWLGAGVAGGSETASTGPTITAGAVTNASEAPTSGSPGSAASPEVTPTSTPNPNNVTQNKTTPTEPASPPTTPKPTSTPKSPPTSTPDPKPKNNTTPAKSGRPTKPPGPVWCDRRDPLARYGSRVQIRCRFRNSTRMEFRLQIWRYSMGPSPPIAPAPDLEEVLTNITAPPGGLLVYDSAPNLTDPHVLWAEGAGPGADPPLYSVTGPLPTQRLIIGEVTPATQGMYYLAWGRMDSPHEYGTWVRVRMFRPPSLTLQPHAVMEGQPFKATCTAAAYYPRNPVEFDWFEDDRQVFNPGQIDTQTHEHPDGFTTVSTVTSEAVGGQVPPRTFTCQMTWHRDSVTFSRRNATGLALVLPRPTITMEFGVRHVVCTAGCVPEGVTFAWFLGDDPSPAAKSAVTAQESCDHPGLATVRSTLPISYDYSEYICRLTGYPAGIPVLEHHGSHQPPPRDPTERQVIEAIEWVGIGIGVLAAGVLVVTAIVYVVRTSQSRQRHR R.

In another embodiment, the HSV-1 gC encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in any of the following GenBankAccession Numbers: AAA45779.1, AAA96680.1, ABI63505.1, ABM52973.1,ABM52976.1, ABM52977.1, ACM62267.1, ADD60042.1, ADD60119.1, ADM22367.1,ADM22444.1, ADM22520.1, ADM22597.1, ADM22674.1, ADM22751.1, ADM22827.1,ADM22904.1, ADM22981.1, ADM23057.1, ADM23133.1, ADM23210.1, ADM23287.1,ADM23361.1, ADM23435.1, ADM23509.1, ADM23583.1, ADM23658.1, ADM23733.1,ADM23809.1, AEQ77075.1, AEQ77099.1, AER37628.1, AER37697.1, AER37767.1,AER37838.1, AER37910.1, AER37981.1, AER38051.2, AFA36179.1, AFA36180.1,AFA36181.1, AFA36182.1, AFA36183.1, AFA36184.1, AFA36185.1, AFA36186.1,AFA36187.1, AFA36188.1, AFA36189.1, AFA36190.1, AFA36191.1, AFA36192.1,AFA36193.1, AFA36194.1, AFA36195.1, AFA36196.1, AFA36197.1, AFA36198.1,AFA36199.1, AFA36200.1, AFA36201.1, AFA36202.1, AFA36203.1, AFE62872.1,AFH78104.1, AFI23635.1, AFK50391.1, AFP86408.1, AGZ01906.1, AIR95840.1,AJE59989.1, AJE60060.1, AJE60131.1, AJE60202.1, AKE48623.1, AKE98415.1,AKE98416.1, AKE98417.1, AKE98418.1, AKE98419.1, AKE98420.1, AKE98421.1,AKE98422.1, AKE98423.1, AKE98424.1, AKE98425.1, AKE98426.1, AKE98427.1,AKE98428.1, AKE98429.1, AKE98430.1, AKE98431.1, AKE98432.1, AKE98433.1,AKE98434.1, AKE98435.1, AKG59227.1, AKG59299.1, AKG59372.1, AKG59444.1,AKG59516.1, AKG59591.1, AKG59663.1, AKG59736.1, AKG59807.1, AKG59879.1,AKG59953.1, AKG60027.1, AKG60099.1, AKG60170.1, AKG60243.1, AKG60316.1,AKG60386.1, AKG60456.1, AKG60528.1, AKG60601.1, AKG60674.1, AKG60745.1,AKG60817.1, AKG60887.1, AKG60959.1, AKG61032.1, AKG61104.1, AKG61175.1,AKG61248.1, AKG61321.1, AKG61392.1, AKG61464.1, AKG61537.1, AKG61611.1,AKG61684.1, AKG61756.1, AKG61828.1, AKG61902.1, AKG61974.1, AKH80444.1,AKH80517.1, AKM76368.1, ALM22613.1, ALM22687.1, ALM22761.1, ALM22835.1,ALO18641.1, ALO18717.1, AMB65642.1, AMB65715.1, AMB65862.1, AMN09813.1,ANN83942.1, ANN84019.1, ANN84095.1, ANN84172.1, ANN84249.1, ANN84326.1,ANN84403.1, ANN84478.1, ANN84555.1, ANN84632.1, ANN84708.1, ANN84785.1,ANN84861.1, ANN84938.1, ANN85014.1, ANN85091.1, ANN85167.1, ANN85242.1,ANN85319.1, ANN85396.1, ANN85472.1, ANN85549.1, ANN85626.1, ANN85703.1,ANN85779.1, AOY34308.1, AOY36663.1, AOY36687.1, ARB08935.1, ARO38059.1,ARO38060.1, ARO38061.1, ARO38062.1, ARO38063.1, ARO38064.1, ARO38065.1,ARO38066.1, ASM47642.1, ASM47719.1, ASM47796.1, ASM47871.1, BAM73394.1,CAA32294.1, CAB40083.1, CAD13356.1, CAD13357.1, CAD13358.1, CAD13359.1,CAD13360.1, CAD13361.1, CAD13362.1, CAD13363.1, CAD13364.1, CAD13365.1,CAD13366.1, CAD13367.1, CAD13368.1, CAD13369.1, CAD13370.1, CAD13371.1,CAD13372.1, CAD13373.1, CAD13374.1, CAD13375.1, CAD13376.1, CAD13377.1,CAD13378.1, P04290.1, P04488.1, P09855.1, P10228.1, P28986.1,SBO07729.1, SBO07793.1, SBO07798.1, SBO07812.1, SBO07880.1, SBS69375.1,SBS69379.1, SBS69440.1, SBS69448.1, SBS69560.1, SBS69599.1, SBS69602.1,SBS69637.1, SBS69790.1, SBT69374.1, SCL76887.1, YP_009137119.1, orYP_009137143.1.

In another embodiment, the composition comprises a modified mRNAencoding an HSV-2 gC protein. In another embodiment, the compositioncomprises a modified mRNA encoding a fragment of an HSV-2 gC protein.

In one embodiment, the nucleotide sequence of the modified mRNA encodingan HSV-2 gC fragment comprises:

(SEQ ID NO: 10) GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGC AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACUCCGCCUCCCCCGGCCGCACCAUCACCGUGGGCCCCCGCGGCAACGCCUCCAACGCCGCCCCCUCCGCCUCCCCCCGCAACGCCUCCGCCCCCCGCACCACCCCCACCCCCCCCCAGCCCCGCAAGGCCACCAAGUCCAAGGCCUCCACCGCCAAGCCCGCCCCCCCCCCCAAGACCGGCCCCCCCAAGACCUCCUCCGAGCCCGUGCGCUGCAACCGCCACGACCCCCUGGCCCGCUACGGCUCCCGCGUGCAGAUCCGCUGCCGCUUCCCCAACUCCACCCGCACCGAGUUCCGCCUGCAGAUCUGGCGCUACGCCACCGCCACCGACGCCGAGAUCGGCACCGCCCCCUCCCUGGAGGAGGUGAUGGUGAACGUGUCCGCCCCCCCCGGCGGCCAGCUGGUGUACGACUCCGCCCCCAACCGCACCGACCCCCACGUGAUCUGGGCCGAGGGCGCCGGCCCCGGCGCCUCCCCCCGCCUGUACUCCGUGGUGGGCCCCCUGGGCCGCCAGCGCCUGAUCAUCGAGGAGCUGACCCUGGAGACCCAGGGCAUGUACUACUGGGUGUGGGGCCGCACCGACCGCCCCUCCGCCUACGGCACCUGGGUGCGCGUGCGCGUGUUCCGCCCCCCCUCCCUGACCAUCCACCCCCACGCCGUGCUGGAGGGCCAGCCCUUCAAGGCCACCUGCACCGCCGCCACCUACUACCCCGGCAACCGCGCCGAGUUCGUGUGGUUCGAGGACGGCCGCCGCGUGUUCGACCCCGCCCAGAUCCACACCCAGACCCAGGAGAACCCCGACGGCUUCUCCACCGUGUCCACCGUGACCUCCGCCGCCGUGGGCGGCCAGGGCCCCCCCCGCACCUUCACCUGCCAGCUGACCUGGCACCGCGACUCCGUGUCCUUCUCCCGCCGCAACGCCUCCGGCACCGCCUCCGUGCUGCCCCGCCCCACCAUCACCAUGGAGUUCACCGGCGACCACGCCGUGUGCACCGCCGGCUGCGUGCCCGAGGGCGUGACCUUCGCCUGGUUCCUGGGCGACGACUCCUCCCCCGCCGAGAAGGUGGCCGUGGCCUCCCAGACCUCCUGCGGCCGCCCCGGCACCGCCACCAUCCGCUCCACCCUGCCCGUGUCCUACGAGCAGACCGAGUACAUCUGCCGCCUGGCCGGCUACCCCGACGGCAUCCCCGUGCUGGAGCACCACUAACUAGUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC.

In one embodiment, all uridine residues are 1-methyl-pseudouridine. Inone embodiment, underlined residues represent 5′ untranslated sequences.In one embodiment, bold residues represent a signal sequence (leadersequence) to assist expression of the gC2 fragment. In one embodiment,italicized residues represent 3′ untranslated sequences and polyadenylation tail.

In another embodiment, the nucleotide sequence of the modified mRNAencoding an HSV-2 gC fragment lacks the 5′ untranslated sequences, thesignal sequence, the 3′ untranslated sequences, the poly adenylationtail, or a combination thereof.

In one embodiment, the HSV-2 gC fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises amino acids 27-426 of gC from HSV-2 strain 333, as set forthin the following amino acid sequence:

(SEQ ID NO: 11) ASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTESRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHH.

In one embodiment, the full length HSV-2 gC encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises the following amino acid sequence:

(SEQ ID NO: 12) MALGRVGLAVGLWGLLWVGVVVVLANASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPKTGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSLEEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEELTLETQGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTWHRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEKVAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHHGSHQPPPRDPTERQVIRAVEGAGIGVAVLVAVVLAGTAVVYLTHASSVRYRRLR.

In another embodiment, the HSV-2 gC encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in any of the following GenBankAccession Numbers: AAA20532.1, AAA66442.1, AAB60549.1, AAB60550.1,AAB60551.1, AAB72101.1, ABU45429.1, ABU45430.1, ABU45431.1, ABU45432.1,ABU45459.1, ABU45460.1, AEV91348.1, AEV91383.1, AEV91407.1, AFM93864.1,AHG54708.1, AKC42808.1, AKC59285.1, AKC59357.1, AKC59428.1, AKC59499.1,AKC59570.1, AMB66008.1, AMB66079.1, AMB66151.1, AMB66224.1, AMB66252.1,AMB66253.1, AMB66368.1, AMB66441.1, AQZ55735.2, AQZ55806.1, AQZ55877.1,AQZ55948.1, AQZ56019.1, AQZ56090.1, AQZ56161.2, AQZ56232.2, AQZ56303.2,AQZ56374.2, AQZ56445.1, AQZ56516.1, AQZ56587.1, AQZ56658.1, AQZ56729.2,AQZ56800.1, AQZ56871.1, AQZ56942.2, AQZ57013.1, AQZ57084.2, AQZ57155.1,AQZ57226.1, AQZ57297.1, AQZ57368.1, AQZ57439.1, AQZ57510.1, AQZ57581.1,AQZ57652.1, AQZ57723.1, AQZ57794.2, AQZ57865.2, AQZ57936.1, AQZ58007.2,AQZ58078.1, AQZ58149.2, AQZ58220.1, AQZ58291.1, AQZ58362.1, AQZ58433.1,AQZ58504.1, AQZ58575.1, AQZ58646.1, AQZ58717.2, AQZ58788.2, AQZ58859.2,AQZ58930.1, AQZ59001.2, AQZ59072.1, AQZ59143.1, ARO38067.1, ARO38068.1,ARO38069.1, ARO38070.1, ARO38071.1, ARO38072.1, CAA25687.1, CAA26025.1,CAB06730.1, CAB06734.1, CAB96544.1, P03173.1, P06475.1, P89475.1,Q89730.1, YP_009137161.1, YP_009137196.1, or YP_009137220.1.

In another embodiment, the gC protein fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises a properdin interfering domain “Properdin-interfering domain”refers, in one embodiment, to a domain that blocks or inhibits bindingof a host C3b molecule with a host properdin molecule. In anotherembodiment, the term refers to a domain that blocks or inhibits aninteraction of a host C3b molecule with a host properdin molecule.

In another embodiment, the gC protein fragment encoded by modified mRNAutilized in the methods and compositions of the present invention is aC5 interfering domain. In another embodiment, the gC protein fragment isa portion of a C5 interfering domain. “C5-interfering domain” refers, inanother embodiment, to a domain that interferes with binding of a hostC3b molecule with a host C5 molecule. In another embodiment, the termrefers to a domain that interferes with the interaction of a host C3bmolecule with a host C5 molecule.

Each modified mRNA encoding gC-1 or gC-2 protein or fragment thereofrepresents a separate embodiment of the present invention.

In another embodiment, a gC protein fragment encoded by modified mRNAutilized in the methods and compositions of the present invention is animmunogenic fragment. In another embodiment, a gC immunoprotectiveantigen need not be the entire protein. The protective immune responsegenerally involves, in another embodiment, an antibody response. Inanother embodiment, mutants, sequence conservative variants, andfunctional conservative variants of gC are useful in methods andcompositions of the present invention, provided that all such variantsretain the required immuno-protective effect. In another embodiment, theimmunogenic fragment can comprise an immuno-protective gC antigen fromany strain of HSV. In another embodiment, the immunogenic fragment cancomprise sequence variants of HSV, as found in infected individuals.

Glycoprotein E

In another embodiment, a composition of the present invention comprisesa modified mRNA encoding HSV-1 gE protein. In another embodiment, thecomposition comprises a modified mRNA encoding a fragment of an HSV-1 gEprotein.

In one embodiment, the nucleotide sequence of the modified mRNA encodingan HSV-1 gD fragment comprises:

(SEQ ID NO: 13) GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGC AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACUCCAAGACCUCCUGGCGCCGCGUGUCCGUGGGCGAGGACGUGUCCCUGCUGCCCGCCCCCGGCCCCACCGGCCGCGGCCCCACCCAGAAGCUGCUGUGGGCCGUGGAGCCCCUGGACGGCUGCGGCCCCCUGCACCCCUCCUGGGUGUCCCUGAUGCCCCCCAAGCAGGUGCCCGAGACCGUGGUGGACGCCGCCUGCAUGCGCGCCCCCGUGCCCCUGGCCAUGGCCUACGCCCCCCCCGCCCCCUCCGCCACCGGCGGCCUGCGCACCGACUUCGUGUGGCAGGAGCGCGCCGCCGUGGUGAACCGCUCCCUGGUGAUCUACGGCGUGCGCGAGACCGACUCCGGCCUGUACACCCUGUCCGUGGGCGACAUCAAGGACCCCGCCCGCCAGGUGGCCUCCGUGGUGCUGGUGGUGCAGCCCGCCCCCGUGCCCACCCCCCCCCCCACCCCCGCCGACUACGACGAGGACGACAACGACGAGGGCGAGGGCGAGGACGAGUCCCUGGCCGGCACCCCCGCCUCCGGCACCCCCCGCCUGCCCCCCUCCCCCGCCCCCCCCCGCUCCUGGCCCUCCGCCCCCGAGGUGUCCCACGUGCGCGGCGUGACCGUGCGCAUGGAGACCCCCGAGGCCAUCCUGUUCUCCCCCGGCGAGGCCUUCUCCACCAACGUGUCCAUCCACGCCAUCGCCCACGACGACCAGACCUACACCAUGGACGUGGUGUGGCUGCGCUUCGACGUGCCCACCUCCUGCGCCGAGAUGCGCAUCUACGAGUCCUGCCUGUACCACCCCCAGCUGCCCGAGUGCCUGUCCCCCGCCGACGCCCCCUGCGCCGCCUCCACCUGGACCUCCCGCCUGGCCGUGCGCUCCUACGCCGGCUGCUCCCGCACCAACCCCCCCCCCCGCUGCUCCGCCGAGGCCCACAUGGAGCCCUUCCCCGGCCUGGCCUGGCAGGCCGCCUCCGUGAACCUGGAGUUCCGCGACGCCUCCCCCCAGCACUCCGGCCUGUACCUGUGCGUGGUGUACGUGAACGACCACAUCCACGCCUGGGGCCACAUCACCAUCAACACCGCCGCCCAGUACCGCAACGCCGUGGUGGAGCAGCCCCUGCCCCAGCGCGGCGCCGACCUGGCCGAGCCCACCCACCCCCACGUGGGCGCCUAACUAGUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC.

In one embodiment, all uridine residues are 1-methyl-pseudouridine. Inone embodiment, underlined residues represent 5′ untranslated sequences.In one embodiment, bold residues represent a signal sequence (leadersequence) to assist expression of the gE1 fragment. In one embodiment,italicized residues represent 3′ untranslated sequences and polyadenylation tail.

In another embodiment, the nucleotide sequence of the modified mRNAencoding an HSV-1 gE fragment lacks the 5′ untranslated sequences, thesignal sequence, the 3′ untranslated sequences, the poly adenylationtail, or a combination thereof.

In one embodiment, the HSV-1 gE fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises amino acids 24-409 of gE from HSV-1 NS strain, as set forth inthe following amino acid sequence:

(SEQ ID NO: 14) KTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGA.

In one embodiment, the gE fragment encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises aminoacids 24-409 of gE from an HSV-1 strain.

In one embodiment, the full length HSV-1 gE encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises the following amino acid sequence:

(SEQ ID NO: 15) MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPTQKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYAPPAPSATGGLRTDFVWQERAAVVNRSLVIYGVRETDSGLYTLSVGDIKDPARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEGEDESLAGTPASGTPRLPPSPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGEAFSTNVSIHAIAHDDQTYTMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAASTWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPFPGLAWQAASVNLEFRDASPQHSGLYLCVVYVNDHIHAWGHITINTAAQYRNAVVEQPLPQRGADLAEPTHPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWRAVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPSTNGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW.

In another embodiment, the HSV-1 gE encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in any of the following GenBankAccession Numbers: AAA45779.1, AAA96680.1, ABI63526.1, ACM62297.1,ADD60055.1, ADD60132.1, ADM22391.1, ADM22468.1, ADM22544.1, ADM22621.1,ADM22698.1, ADM22775.1, ADM22851.1, ADM22928.1, ADM23005.1, ADM23081.1,ADM23157.1, ADM23233.1, ADM23311.1, ADM23385.1, ADM23459.1, ADM23533.1,ADM23607.1, ADM23682.1, ADM23757.1, ADM23833.1, ADN34689.1, ADN34692.1,ADN34695.1, AEQ77099.1, AER37649.1, AER37717.1, AER37788.1, AER37859.1,AER37931.1, AER38002.1, AER38072.1, AFA36179.1, AFA36180.1, AFA36181.1,AFA36182.1, AFA36183.1, AFA36184.1, AFA36185.1, AFA36186.1, AFA36187.1,AFA36188.1, AFA36189.1, AFA36190.1, AFA36191.1, AFA36192.1, AFA36193.1,AFA36194.1, AFA36195.1, AFA36196.1, AFA36197.1, AFA36198.1, AFA36199.1,AFA36200.1, AFA36201.1, AFA36202.1, AFA36203.1, AFE62896.1, AFI23659.1,AFK50417.1, AFP86432.1, AGZ01930.1, AIR95859.1, AJE60011.1, AJE60082.1,AJE60153.1, AJE60224.1, AJE60295.1, AKE48647.1, AKE98373.1, AKE98374.1,AKE98375.1, AKE98376.1, AKE98377.1, AKE98378.1, AKE98379.1, AKE98380.1,AKE98381.1, AKE98382.1, AKE98383.1, AKE98384.1, AKE98385.1, AKE98386.1,AKE98387.1, AKE98388.1, AKE98389.1, AKE98390.1, AKE98391.1, AKE98392.1,AKE98393.1, AKG59248.1, AKG59320.1, AKG59393.1, AKG59464.1, AKG59538.1,AKG59611.1, AKG59684.1, AKG59757.1, AKG59828.1, AKG59900.1, AKG59974.1,AKG60048.1, AKG60120.1, AKG60191.1, AKG60263.1, AKG60336.1, AKG60406.1,AKG60476.1, AKG60548.1, AKG60622.1, AKG60694.1, AKG60765.1, AKG60837.1,AKG60908.1, AKG60980.1, AKG61052.1, AKG61125.1, AKG61196.1, AKG61269.1,AKG61341.1, AKG61413.1, AKG61486.1, AKG61558.1, AKG61631.1, AKG61705.1,AKG61776.1, AKG61849.1, AKG61922.1, AKG61995.1, AKH80465.1, AKH80538.1,ALM22637.1, ALM22711.1, ALM22785.1, ALM22859.1, ALO18664.1, ALO18740.1,AMB65664.1, AMB65737.1, AMB65811.1, AMB65887.1, AMB65958.1, AMN09834.1,ANN83966.1, ANN84043.1, ANN84119.1, ANN84196.1, ANN84273.1, ANN84350.1,ANN84426.1, ANN84502.1, ANN84579.1, ANN84655.1, ANN84732.1, ANN84808.1,ANN84885.1, ANN84961.1, ANN85038.1, ANN85114.1, ANN85189.1, ANN85266.1,ANN85343.1, ANN85418.1, ANN85496.1, ANN85573.1, ANN85650.1, ANN85726.1,ANN85803.1, AOY34085.1, AOY36687.1, ARB08959.1, ARO38073.1, ARO38074.1,ARO38075.1, ARO38076.1, ARO38077.1, ARO38078.1, ARO38079.1, ARO38080.1,ASM47642.1, ASM47666.1, ASM47743.1, ASM47820.1, ASM47895.1, BAM73421.1,CAA26062.1, CAA32272.1, CAF24756.1, CAF24757.1, CAF24758.1, CAF24759.1,CAF24760.1, CAF24761.1, CAF24762.1, CAF24763.1, CAF24764.1, CAF24765.1,CAF24766.1, CAF24767.1, CAF24768.1, CAF24769.1, CAF24770.1, CAF24771.1,CAF24772.1, CAF24773.1, CAF24774.1, CAF24775.1, CAF24776.1, CAF24777.1,CAF24778.1, CAF24779.1, CAF24780.1, CAF24781.1, CAF24782.1, CAF24783.1,CAF24784.1, CAF24785.1, P04290.1, P04488.1, P28986.1, Q703F0.1,SBO07910.1, SBS69571.1, SBS69576.1, SBS69595.1, SBS69636.1, SBS69693.1,SBS69701.1, SBS69722.1, SBS69732.1, SBS69813.1, SBT69397.1, orYP_009137143.1.

In another embodiment, the composition comprises a modified mRNAencoding an HSV-2 gE protein. In another embodiment, the compositioncomprises a modified mRNA encoding a fragment of an HSV-2 gE protein.

In one embodiment, the nucleotide sequence of the modified mRNA encodingan HSV-2 gE fragment comprises:

(SEQ ID NO: 16) GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGC AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACUCCCGCACCUCCUGGAAGCGCGUGACCUCCGGCGAGGACGUGGUGCUGCUGCCCGCCCCCGCCGGCCCCGAGGAGCGCACCCGCGCCCACAAGCUGCUGUGGGCCGCCGAGCCCCUGGACGCCUGCGGCCCCCUGCGCCCCUCCUGGGUGGCCCUGUGGCCCCCCCGCCGCGUGCUGGAGACCGUGGUGGACGCCGCCUGCAUGCGCGCCCCCGAGCCCCUGGCCAUCGCCUACUCCCCCCCCUUCCCCGCCGGCGACGAGGGCCUGUACUCCGAGCUGGCCUGGCGCGACCGCGUGGCCGUGGUGAACGAGUCCCUGGUGAUCUACGGCGCCCUGGAGACCGACUCCGGCCUGUACACCCUGUCCGUGGUGGGCCUGUCCGACGAGGCCCGCCAGGUGGCCUCCGUGGUGCUGGUGGUGGAGCCCGCCCCCGUGCCCACCCCCACCCCCGACGACUACGACGAGGAGGACGACGCCGGCGUGUCCGAGCGCACCCCCGUGUCCGUGCCCCCCCCCACCCCCCCCCGCCGCCCCCCCGUGGCCCCCCCCACCCACCCCCGCGUGAUCCCCGAGGUGUCCCACGUGCGCGGCGUGACCGUGCACAUGGAGACCCCCGAGGCCAUCCUGUUCGCCCCCGGCGAGACCUUCGGCACCAACGUGUCCAUCCACGCCAUCGCCCACGACGACGGCCCCUACGCCAUGGACGUGGUGUGGAUGCGCUUCGACGUGCCCUCCUCCUGCGCCGAGAUGCGCAUCUACGAGGCCUGCCUGUACCACCCCCAGCUGCCCGAGUGCCUGUCCCCCGCCGACGCCCCCUGCGCCGUGUCCUCCUGGGCCUACCGCCUGGCCGUGCGCUCCUACGCCGGCUGCUCCCGCACCACCCCCCCCCCCCGCUGCUUCGCCGAGGCCCGCAUGGAGCCCGUGCCCGGCCUGGCCUGGCUGGCCUCCACCGUGAACCUGGAGUUCCAGCACGCCUCCCCCCAGCACGCCGGCCUGUACCUGUGCGUGGUGUACGUGGACGACCACAUCCACGCCUGGGGCCACAUGACCAUCUCCACCGCCGCCCAGUACCGCAACGCCGUGGUGGAGCAGCACCUGCCCCAGCGCCAGCCCGAGCCCGUGGAGCCCACCCGCCCCCACGUGCGCGCCUAACUAGUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAC

In one embodiment, all uridine residues are 1-methyl-pseudouridine. Inone embodiment, underlined residues represent 5′ untranslated sequences.In one embodiment, bold residues represent a signal sequence (leadersequence) to assist expression of the gE2 fragment. In one embodiment,italicized residues represent 3′ untranslated sequences and polyadenylation tail.

In another embodiment, the nucleotide sequence of the modified mRNAencoding an HSV-2 gE fragment lacks the 5′ untranslated sequences, thesignal sequence, the 3′ untranslated sequences, the poly adenylationtail, or a combination thereof.

In one embodiment, the HSV-2 gE fragment encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises amino acids 24-405 of gE from HSV-2 strain 2.12 as set forthin the following amino acid sequence:

(SEQ ID NO: 17) RTSWKRVTSGEDVVLLPAPAGPEERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPVPTPTPDDYDEEDDAGVSERTPVSVPPPTPPRRPPVAPPTHPRVIPEVSHVRGVTVHMETPEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCAEMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRA.

In one embodiment, the full length HSV-2 gE encoded by modified mRNAutilized in the methods and compositions of the present inventioncomprises the following amino acid sequence:

(SEQ ID NO: 18) MARGAGLVFFVGVWVVSCLAAAPRTSWKRVTSGEDVVLLPAPAERTRAHKLLWAAEPLDACGPLRPSWVALWPPRRVLETVVDAACMRAPEPLAIAYSPPFPAGDEGLYSELAWRDRVAVVNESLVIYGALETDSGLYTLSVVGLSDEARQVASVVLVVEPAPVPTPTPDDYDEEDDAGVTNARRSAFPPQPPPRRPPVAPPTHPRVIPEVSHVRGVTVHMETLEAILFAPGETFGTNVSIHAIAHDDGPYAMDVVWMRFDVPSSCADMRIYEACLYHPQLPECLSPADAPCAVSSWAYRLAVRSYAGCSRTTPPPRCFAEARMEPVPGLAWLASTVNLEFQHASPQHAGLYLCVVYVDDHIHAWGHMTISTAAQYRNAVVEQHLPQRQPEPVEPTRPHVRAPHPAPSARGPLRLGAVLGAALLLAALGLSAWACMTCWRRRSWRAVKSRASATGPTYIRVADSELYADWSSDSEGERDGSLWQDPPERPDSPSTNGSGFEILSPTAPSVYPHSEGRKSRRPLTTFGSGSPGRRHSQASYPSVLW.

In another embodiment, the HSV-2 gE encoded by modified mRNA utilized inthe methods and compositions of the present invention comprises theamino acid sequences as set forth in any of the following GenBankAccession Numbers: ABU45436.1, ABU45437.1, ABU45438.1, ABU45439.1,ABW83306.1, ABW83308.1, ABW83310.1, ABW83312.1, ABW83314.1, ABW83316.1,ABW83318.1, ABW83320.1, ABW83322.1, ABW83324.1, ABW83326.1, ABW83328.1,ABW83330.1, ABW83332.1, ABW83334.1, ABW83336.1, ABW83338.1, ABW83340.1,ABW83342.1, ABW83344.1, ABW83346.1, ABW83348.1, ABW83350.1, ABW83352.1,ABW83354.1, ABW83356.1, ABW83358.1, ABW83360.1, ABW83362.1, ABW83364.1,ABW83366.1, ABW83368.1, ABW83370.1, ABW83372.1, ABW83374.1, ABW83376.1,ABW83378.1, ABW83380.1, ABW83382.1, ABW83384.1, ABW83386.1, ABW83388.1,ABW83390.1, ABW83392.1, ABW83394.1, ABW83396.1, ABW83398.1, ABW83400.1,ABZ04069.1, AEV91407.1, AHG54732.1, AKC42830.1, AKC59307.1, AKC59378.1,AKC59449.1, AKC59520.1, AKC59591.1, AMB66104.1, AMB66173.1, AMB66246.1,AMB66465.1, AQZ55756.1, AQZ55827.1, AQZ55898.1, AQZ55969.2, AQZ56040.2,AQZ56111.2, AQZ56182.1, AQZ56253.2, AQZ56324.1, AQZ56395.1, AQZ56466.2,AQZ56537.1, AQZ56608.1, AQZ56679.1, AQZ56750.1, AQZ56821.2, AQZ56892.1,AQZ56963.2, AQZ57034.2, AQZ57105.1, AQZ57176.1, AQZ57247.2, AQZ57318.2,AQZ57389.2, AQZ57460.2, AQZ57531.2, AQZ57602.2, AQZ57673.1, AQZ57744.2,AQZ57815.1, AQZ57886.1, AQZ57957.2, AQZ58028.2, AQZ58099.1, AQZ58170.2,AQZ58241.2, AQZ58312.2, AQZ58383.2, AQZ58454.2, AQZ58525.2, AQZ58596.1,AQZ58667.1, AQZ58738.2, AQZ58809.2, AQZ58880.2, AQZ58951.2, AQZ59022.2,AQZ59093.1, AQZ59164.1, ARO38081.1, ARO38082.1, ARO38083.1, ARO38084.1,ARO38085.1, ARO38086.1, CAB06715.1, P89436.1, P89475.1, orYP_009137220.1.

In another embodiment, a gE fragment encoded by modified mRNA utilizedin the methods and compositions of the present invention comprises anIgG Fc-binding domain of the gE protein. In another embodiment, the gEdomain encoded by modified mRNA utilized in the methods and compositionsof the present invention is any other gE domain known in the art tomediate binding to IgG Fc.

In another embodiment, the gE protein encoded by modified mRNA utilizedin the methods and compositions of the present invention comprises a gEdomain involved in cell-to-cell spread.

In another embodiment, the gE fragment encoded by modified mRNA fragmentutilized in the methods and compositions of the present inventioncomprises an immune evasion domain. In another embodiment, the gEfragment encoded by modified mRNA fragment utilized in the methods andcompositions of the present invention comprises a portion of an immuneevasion domain.

Each modified mRNA encoding gE-1 or gE-2 protein or fragment thereofrepresents a separate embodiment of the present invention.

In another embodiment, a gE protein fragment encoded by modified mRNAutilized in the methods and compositions of the present invention is animmunogenic fragment. In another embodiment, a gE immunoprotectiveantigen need not be the entire protein. The protective immune responsegenerally involves, in another embodiment, an antibody response. Inanother embodiment, mutants, sequence conservative variants, andfunctional conservative variants of gE are useful in methods andcompositions of the present invention, provided that all such variantsretain the required immuno-protective effect. In another embodiment, theimmunogenic fragment can comprise an immuno-protective gE antigen fromany strain of HSV. In another embodiment, the immunogenic fragment cancomprise sequence variants of HSV, as found in infected individuals.

In one embodiment, an HSV glycoprotein encoded by modified mRNA utilizedin the methods and compositions of the present invention is a homologueof the sequence provided herein. In another embodiment, an HSVglycoprotein encoded by modified mRNA utilized in the methods andcompositions of the present invention is an isoform of the sequenceprovided herein. In another embodiment, an HSV glycoprotein encoded bymodified mRNA utilized in the methods and compositions of the presentinvention is a variant of the sequence provided herein. In anotherembodiment, an HSV glycoprotein encoded by modified mRNA utilized in themethods and compositions of the present invention is a fragment of thesequence provided herein.

In another embodiment, the glycoprotein fragment encoded by modifiedmRNA of the methods and compositions of the present invention comprisesthe ectodomain of the glycoprotein. In another embodiment, theglycoprotein fragment encoded by modified mRNA of the methods andcompositions of the present invention consists of the ectodomain of theglycoprotein. In another embodiment, the glycoprotein fragment encodedby modified mRNA of the methods and compositions of the presentinvention comprises a fragment of the ectodomain of the glycoprotein. Inanother embodiment, the glycoprotein fragment may be any glycoproteinfragment known in the art.

In another embodiment, the glycoprotein or immunogenic fragment encodedby modified mRNA fragment utilized in the methods and compositions ofthe present invention may be from any strain of HSV. In anotherembodiment, the immunogenic fragment encoded by modified mRNA fragmentutilized in the methods and compositions of the present invention maycomprise sequence variants of HSV, as found in infected individuals.

In one embodiment, “variant” refers to an amino acid or nucleic acidsequence (or in other embodiments, an organism or tissue) that isdifferent from the majority of the population but is still sufficientlysimilar to the common mode to be considered to be one of them, forexample splice variants. In one embodiment, the variant may a sequenceconservative variant, while in another embodiment, the variant may be afunctional conservative variant. In one embodiment, a variant maycomprise an addition, deletion or substitution of one or more aminoacids.

“Immune evasion domain” refers, in one embodiment, to a domain thatinterferes with or reduces in vivo anti-HSV efficacy of anti-HSVantibodies (e.g. anti-gD antibodies). In another embodiment, the domaininterferes or reduces in vivo anti-HSV efficacy of an anti-HSV immuneresponse. In another embodiment, the domain reduces the immunogenicityof an HSV protein (e.g. gD) during subsequent infection. In anotherembodiment, the domain reduces the immunogenicity of an HSV proteinduring subsequent challenge. In another embodiment, the domain reducesthe immunogenicity of HSV during subsequent challenge. In anotherembodiment, the domain reduces the immunogenicity of an HSV protein inthe context of ongoing HSV infection. In another embodiment, the domainreduces the immunogenicity of HSV in the context of ongoing HSVinfection. In another embodiment, the domain functions as an IgG Fcreceptor. In another embodiment, the domain promotes antibody bipolarbridging, which in one embodiment, is a term that refers to an antibodymolecule binding by its Fab domain to an HSV antigen and by its Fcdomain to a separate HSV antigen, such as in one embodiment, gE, therebyblocking the ability of the Fc domain to activate complement.

The present invention also provides for modified mRNA encoding analogsof HSV proteins or polypeptides, or fragments thereof. Analogs maydiffer from naturally occurring proteins or peptides by conservativeamino acid sequence substitutions or by modifications which do notaffect sequence, or by both.

In another embodiment, an HSV glycoprotein encoded by modified mRNA ofthe present invention is homologous to a sequence set forth hereinabove,either expressly or by reference to a GenBank entry. The terms“homology,” “homologous,” etc, when in reference to any protein orpeptide, refer, in one embodiment, to a percentage of amino acidresidues in the candidate sequence that are identical with the residuesof a corresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Methods and computer programs for the alignment arewell known in the art.

In another embodiment, “homology” refers to identity of a proteinsequence encoded by a modified mRNA to a sequence disclosed herein ofgreater than 70%. In another embodiment, the identity is greater than72%. In another embodiment, the identity is greater than 75%. In anotherembodiment, the identity is greater than 78%. In another embodiment, theidentity is greater than 80%. In another embodiment, the identity isgreater than 82%. In another embodiment, the identity is greater than83%. In another embodiment, the identity is greater than 85%. In anotherembodiment, the identity is greater than 87%. In another embodiment, theidentity is greater than 88%. In another embodiment, the identity isgreater than 90%. In another embodiment, the identity is greater than92%. In another embodiment, the identity is greater than 93%. In anotherembodiment, the identity is greater than 95%. In another embodiment, theidentity is greater than 96%. In another embodiment, the identity isgreater than 97%. In another embodiment, the identity is greater than98%. In another embodiment, the identity is greater than 99%. In anotherembodiment, the identity is 100%.

In one embodiment, “isoform” refers to a version of a molecule, forexample, a protein, with only slight differences to another isoform ofthe same protein. In one embodiment, isoforms may be produced fromdifferent but related genes, or in another embodiment, may arise fromthe same gene by alternative splicing. In another embodiment, isoformsare caused by single nucleotide polymorphisms.

In another embodiment, the modified mRNA encoding a glycoprotein orglycoprotein fragment as described herein further encodes an antigenictag. In one embodiment, the tag is a histidine (“His”) tag. In oneembodiment, the His tag comprises 5 histidine residues. In anotherembodiment, the His tag comprises 6 histidine residues.

In another embodiment, methods and compositions of the present inventionutilize a chimeric molecule, comprising a fusion of a modified mRNAencoding an HSV protein with a modified mRNA encoding a tag polypeptidethat provides an epitope to which an anti-tag antibody can selectivelybind. The epitope tag is placed, in other embodiments, at the amino- orcarboxyl-terminus of the protein or in an internal location therein. Thepresence of such epitope-tagged forms of the recombinant HSV protein isdetected, in another embodiment, using an antibody against the tagpolypeptide. In another embodiment, inclusion of the epitope tag enablesthe recombinant HSV protein to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are known in the art.

In one embodiment, the compositions of the present invention comprise anadjuvant, while in another embodiment, the compositions do not comprisean adjuvant. “Adjuvant” refers, in another embodiment, to compoundsthat, when administered to an individual or tested in vitro, increasethe immune response to an antigen in the individual or test system towhich the antigen is administered. In another embodiment, an immuneadjuvant enhances an immune response to an antigen that is weaklyimmunogenic when administered alone, i.e., inducing no or weak antibodytiters or cell-mediated immune response. In another embodiment, theadjuvant increases antibody titers to the antigen. In anotherembodiment, the adjuvant lowers the dose of the antigen effective toachieve an immune response in the individual. Multiple types ofadjuvants are known in the art and described in detail in U. S. PatentPublication 2013/0028925 which is hereby incorporated by referenceherein.

Modified mRNAs

In one embodiment, the present invention provides compositionscomprising modified mRNAs and methods of use thereof. In one embodiment,the modified mRNA comprises one or more modified nucleoside residues.

In another embodiment, the modified nucleoside of the methods andcompositions of the present invention is m5C (5-methylcytidine). Inanother embodiment, the modified nucleoside is m5U (5-methyluridine). Inanother embodiment, the modified nucleoside is m6A (N6-methyladenosine).In another embodiment, the modified nucleoside is s2U (2-thiouridine).In another embodiment, the modified nucleoside is Ψ (pseudouridine). Inanother embodiment, the modified nucleoside is Um (2′-O-methyluridine).

In other embodiments, the modified nucleoside is m¹A(1-methyladenosine), m²A (2-methyladenosine), m⁶A (N6-methyladenosine),Am (2′-O-methyladenosine), ms²m⁶A (2-methylthio-N6-methyladenosine), i⁶A(N6-isopentenyladenosine), ms²i⁶A(2-methylthio-N6-isopentenyladenosine), io⁶A(N6-(cis-hydroxyisopentenyl)adenosine), ms²io⁶A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g⁶A(N6-glycinylcarbamoyladenosine), t⁶A (N6-threonylcarbamoyladenosine),ms²t⁶A (2-methylthio-N6-threonyl carbamoyladenosine), m⁶t⁶A(N6-methyl-N6-threonylcarbamoyladenosine), hn⁶A(N6-hydroxynorvalylcarbamoyladenosine), ms²hn⁶A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine), Ar(p)(2′-O-ribosyladenosine (phosphate)), I (inosine), m¹I (1-methylinosine),m¹Im (1,2′-O-dimethylinosine), m³C (3-methylcytidine), m⁵C(5-methylcytidine), Cm (2′-O-methylcytidine), s²C (2-thiocytidine), ac⁴C(N4-acetylcytidine), f⁵C (5-formylcytidine), m⁵Cm(5,2′-O-dimethylcytidine), ac⁴Cm (N4-acetyl-2′-O-methylcytidine), k²C(lysidine), m¹G (1-methylguanosine), m²G (N2-methylguanosine), m⁷G(7-methylguanosine), Gm (2′-O-methylguanosine), m² ₂G(N2,N2-dimethylguanosine), m²Gm (N2,2′-O-dimethylguanosine), m² ₂Gm(N2,N2,2′-O-trimethylguanosine), Gr(p) (2′-O-ribosylguanosine(phosphate)), yW (wybutosine), o₂yW (peroxywybutosine), OHyW(hydroxywybutosine), OHyW* (undermodified hydroxywybutosine), imG(wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine),galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQ0(7-cyano-7-deazaguanosine), preQ1 (7-aminomethyl-7-deazaguanosine), G⁺(archaeosine), Ψ (pseudouridine), D (dihydrouridine), m⁵U(5-methyluridine), Um (2′-O-methyluridine), m⁵Um(5,2′-O-dimethyluridine), m¹Ψ (1-methylpseudouridine), Ψm(2′-O-methylpseudouridine), s²U (2-thiouridine), s⁴U (4-thiouridine),m⁵s²U (5-methyl-2-thiouridine), s²Um (2-thio-2′-O-methyluridine), acp³U(3-(3-amino-3-carboxypropyl)uridine), ho⁵U (5-hydroxyuridine), mo⁵U(5-methoxyuridine), cmo⁵U (uridine 5-oxyacetic acid), mcmo⁵U (uridine5-oxyacetic acid methyl ester), chm⁵U (5-(carboxyhydroxymethyl)uridine),mchm⁵U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm⁵U(5-methoxycarbonylmethyluridine), mcm⁵Um(5-methoxycarbonylmethyl-2′-O-methyluridine), mcm⁵s²U(5-methoxycarbonylmethyl-2-thiouridine), nm⁵s²U(5-aminomethyl-2-thiouridine), mnm⁵U (5-methylaminomethyluridine),mnm⁵s²U (5-methylaminomethyl-2-thiouridine), mnm⁵se²U(5-methylaminomethyl-2-selenouridine), ncm⁵U (5-carbamoylmethyluridine),ncm⁵Um (5-carbamoylmethyl-2′-O-methyluridine), cmnm⁵U(5-carboxymethylaminomethyluridine), cmnm⁵Um(5-carboxymethylaminomethyl-2′-O-methyluridine), cmnm⁵s²U(5-carboxymethylaminomethyl-2-thiouridine), m⁶ ₂A(N6,N6-dimethyladenosine), Im (2′-O-methylinosine), m⁴C(N4-methylcytidine), m⁴Cm (N4,2′-O-dimethylcytidine), hm⁵C(5-hydroxymethylcytidine), m³U (3-methyluridine), m¹acp³Ψ(1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine), cm⁵U(5-carboxymethyluridine), m⁶Am (N6,2′-O-dimethyladenosine), m⁶ ₂Am(N6,N6,2′-O-trimethyladenosine), m^(2,7)G (N2,7-dimethylguanosine),m^(2,2,7)G (N2,N2,7-trimethylguanosine), m³Um (3,2′-O-dimethyluridine),m⁵D (5-methyldihydrouridine), m³Ψ (3-methylpseudouridine), f⁵Cm(5-formyl-2′-O-methylcytidine), m¹Gm (1,2′-O-dimethylguanosine), m¹Am(1,2′-O-dimethyladenosine), τm⁵U (5-taurinomethyluridine), τm⁵s²U(5-taurinomethyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2(isowyosine), ac⁶A (N6-acetyladenosine), inm⁵U(5-(isopentenylaminomethyl)uridine), inm⁵s²U(5-(isopentenylaminomethyl)-2-thiouridine), inm⁵Um(5-(isopentenylaminomethyl)-2′-O-methyluridine), m^(2,7)Gm(N2,7,2′-O-trimethylguano sine), m⁴ ₂Cm (N4,N4,2′-O-trimethylcytidine),C⁺ (agmatidine), m⁸A (8-methyladenosine), gmnm⁵s²U (geranylated5-methylaminomethyl-2-thiouridine), gcmnm⁵s²U (geranylated5-carboxymethylaminomethyl-2-thiouridine), or cnm⁵U(5-cyanomethyl-uridine).

In one embodiment, the modified nucleoside residues are pseudouridine orpseudouridine family residues.

In one embodiment, the modified mRNA comprises pseudouridine residues.In one embodiment, pseudouridine refers to the C-glycoside isomer of thenucleoside uridine. In one embodiment, pseudouridine residues comprisem¹acp³Ψ (1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, m³Ψ(1-methylpseudouridine), Ψm (2′-O-methylpseudouridine, m⁵D(5-methyldihydrouridine), m³Ψ (3-methylpseudouridine), or a combinationthereof. In one embodiment, said pseudouridine residues comprise1-methylpseudouridine residues instead of uridine.

In one embodiment, the modified nucleoside residues are pseudouridineanalogues. In one embodiment, a “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹Ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴Ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³Ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³Ψ), and2′-O-methyl-pseudouridine (Ψm).

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (Ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τcm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τrm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methylpseudouridine (m¹Ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴Ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³′),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as1-methylpseudouridine (m¹Ψ), 3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³Ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (Ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-β-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-β-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adeno sine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac^(6A)), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-β-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adeno sine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQo),7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guano sine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²,7G),N2,N2,7-dimethyl-guanosine (m², 2,7G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m²⁷Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m¹Im), and 2′-O-ribosylguanosine (phosphate)(Gr(p)).

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modifications on the Internucleoside Linkage

The modified nucleotides, which may be incorporated into apolynucleotide, primary construct, or mRNA molecule, can be modified onthe internucleoside linkage (e.g., phosphate backbone). Herein, in thecontext of the polynucleotide backbone, the phrases “phosphate” and“phosphodiester” are used interchangeably. Backbone phosphate groups canbe modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the wholesale replacement of an unmodified phosphate moietywith another internucleoside linkage as described herein. Examples ofmodified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenates, boranophosphates, boranophosphateesters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates,alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioateshave both non-linking oxygens replaced by sulfur. The phosphate linkercan also be modified by the replacement of a linking oxygen withnitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates),and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked polynucleotides, primaryconstructs, or mmRNA molecules are expected to also reduce the innateimmune response through weaker binding/activation of cellular innateimmune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adeno sine,5′-O-(1-thiophosphate)-cytidine (a-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The polynucleotides, primary constructs, and mmRNA of the invention caninclude a combination of modifications to the sugar, the nucleobase,and/or the internucleoside linkage.

In another embodiment, the purified preparation of RNA,oligoribonucleotide, or polyribonucleotide of the methods andcompositions of the present invention comprises a combination of two ormore of the above-described modifications. In another embodiment, thepurified preparation of the RNA or oligoribonucleotide comprises acombination of three or more of the above-described modifications. Inanother embodiment, the purified preparation of the RNA oroligoribonucleotide comprises a combination of more than three of theabove-described modifications.

In one embodiment, the modified mRNAs comprise in vitro-synthesizedmodified mRNAs.

In one embodiment, the present invention comprises one or more modifiedmRNAs encoding an HSV glycoprotein. In one embodiment, the modified RNAcomprises pseudouridine or pseudouridine family residues. In anotherembodiment, the modified mRNAs of the present invention are capable ofdirecting protein expression of HSV glycoproteins encoded thereon.

In another embodiment, the present invention provides an invitro-transcribed mRNA molecule encoding an HSV glycoprotein, comprisinga pseudouridine. In another embodiment, the present invention provides asynthetic mRNA molecule encoding an HSV glycoprotein, comprising apseudouridine.

In another embodiment, an in vitro-transcribed mRNA molecule of themethods and compositions of the present invention is synthesized by T7phage RNA polymerase. In another embodiment, the molecule is synthesizedby SP6 phage RNA polymerase. In another embodiment, the molecule issynthesized by T3 phage RNA polymerase. In another embodiment, themolecule is synthesized by a polymerase selected from the abovepolymerases. In another embodiment, the mRNA is synthesized chemicallyon a column similar to DNA.

In another embodiment, the nucleoside that is modified in an RNA,oligoribonucleotide, or polyribonucleotide of the methods andcompositions of the present invention is uridine (U). In anotherembodiment, the modified nucleoside is cytidine (C). In anotherembodiment, the modified nucleoside is adenine (A). In anotherembodiment the modified nucleoside is guanine (G).

In another embodiment, the modified mRNA of the methods and compositionsof the present invention further comprises a poly-A tail. In anotherembodiment, the modified mRNA of the methods and compositions of thepresent invention does not comprise a poly-A tail. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the modified mRNA of the methods and compositionsof the present invention comprises an m7GpppG cap. In anotherembodiment, the modified mRNA of the methods and compositions of thepresent invention does not comprise an m7GpppG cap. In anotherembodiment, the modified mRNA of the methods and compositions of thepresent invention comprises a 3′-O-methyl-m7GpppG. In anotherembodiment, the modified mRNA of methods and composition of the presentinvention comprise a non-reversible cap analog, which, in oneembodiment, is added during transcription of the mRNA. In anotherembodiment, the modified mRNA of methods and composition of the presentinvention comprise an anti-reverse cap analog. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the modified mRNA of the methods and compositionsof the present invention further comprises a cap-independenttranslational enhancer. In another embodiment, the modified mRNA of themethods and compositions of the present invention does not comprise acap-independent translational enhancer. In another embodiment, thecap-independent translational enhancer is a tobacco etch virus (TEV)cap-independent translational enhancer. In another embodiment, thecap-independent translational enhancer is any other cap-independenttranslational enhancer known in the art. Each possibility represents aseparate embodiment of the present invention.

In one embodiment, “pseudouridine” refers to m¹acp³Ψ(1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine. In anotherembodiment, the term refers to m¹Ψ (1-methylpseudouridine). In anotherembodiment, the term refers to Ψm (2′-O-methylpseudouridine. In anotherembodiment, the term refers to m⁵D (5-methyldihydrouridine). In anotherembodiment, the term refers to m³Ψ (3-methylpseudouridine). In anotherembodiment, the modified nucleoside is 4′ (pseudouridine). In anotherembodiment, the term refers to a pseudouridine moiety that is notfurther modified. In another embodiment, the term refers to amonophosphate, diphosphate, or triphosphate of any of the abovepseudouridines. In another embodiment, the term refers to any otherpseudouridine known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the modified RNA comprises a modified nucleoside,which in one embodiment, comprises m⁵C, m5U, m⁶A, s²U, Ψ, 2′-O-methyl-U,2′-O-methylpseudouridine, or a combination thereof.

In another embodiment, the present invention provides a method fordelivering a recombinant protein to a subject, the method comprising thestep of contacting the subject with a modified mRNA of the methods andcompositions of the present invention, thereby delivering a recombinantprotein to a subject.

In another embodiment, a method of the present invention comprisesincreasing the number, percentage, or frequency of modified uridinenucleosides in the RNA molecule to decrease immunogenicity or increaseefficiency of translation. In one embodiment, the number of modifieduridine residues in an RNA, oligoribonucleotide, or polyribonucleotidemolecule determines the magnitude of the effects observed in the presentinvention.

In another embodiment, between 0.1% and 100% of the uridine residues inthe modified mRNAs of the methods and compositions of the presentinvention are modified (e.g. by the presence of pseudouridine). Inanother embodiment, 0.1% of the residues are modified. In anotherembodiment, 0.2%. In another embodiment, the fraction is 0.3%. Inanother embodiment, the fraction is 0.4%. In another embodiment, thefraction is 0.5%. In another embodiment, the fraction is 0.6%. Inanother embodiment, the fraction is 0.8%. In another embodiment, thefraction is 1%. In another embodiment, the fraction is 1.5%. In anotherembodiment, the fraction is 2%. In another embodiment, the fraction is2.5%. In another embodiment, the fraction is 3%. In another embodiment,the fraction is 4%. In another embodiment, the fraction is 5%. Inanother embodiment, the fraction is 6%. In another embodiment, thefraction is 8%. In another embodiment, the fraction is 10%. In anotherembodiment, the fraction is 12%. In another embodiment, the fraction is14%. In another embodiment, the fraction is 16%. In another embodiment,the fraction is 18%. In another embodiment, the fraction is 20%. Inanother embodiment, the fraction is 25%. In another embodiment, thefraction is 30%. In another embodiment, the fraction is 35%. In anotherembodiment, the fraction is 40%. In another embodiment, the fraction is45%. In another embodiment, the fraction is 50%. In another embodiment,the fraction is 60%. In another embodiment, the fraction is 70%. Inanother embodiment, the fraction is 80%. In another embodiment, thefraction is 90%. In another embodiment, the fraction is 100%.

In another embodiment, the fraction is less than 5%. In anotherembodiment, the fraction is less than 3%. In another embodiment, thefraction is less than 1%. In another embodiment, the fraction is lessthan 2%. In another embodiment, the fraction is less than 4%. In anotherembodiment, the fraction is less than 6%. In another embodiment, thefraction is less than 8%. In another embodiment, the fraction is lessthan 10%. In another embodiment, the fraction is less than 12%. Inanother embodiment, the fraction is less than 15%. In anotherembodiment, the fraction is less than 20%. In another embodiment, thefraction is less than 30%. In another embodiment, the fraction is lessthan 40%. In another embodiment, the fraction is less than 50%. Inanother embodiment, the fraction is less than 60%. In anotherembodiment, the fraction is less than 70%

In another embodiment, 0.1% of the residues of a given uridinenucleotide are modified. In another embodiment, the fraction of thenucleotide is 0.2%. In another embodiment, the fraction is 0.3%. Inanother embodiment, the fraction is 0.4%. In another embodiment, thefraction is 0.5%. In another embodiment, the fraction is 0.6%. Inanother embodiment, the fraction is 0.8%. In another embodiment, thefraction is 1%. In another embodiment, the fraction is 1.5%. In anotherembodiment, the fraction is 2%. In another embodiment, the fraction is2.5%. In another embodiment, the fraction is 3%. In another embodiment,the fraction is 4%. In another embodiment, the fraction is 5%. Inanother embodiment, the fraction is 6%. In another embodiment, thefraction is 8%. In another embodiment, the fraction is 10%. In anotherembodiment, the fraction is 12%. In another embodiment, the fraction is14%. In another embodiment, the fraction is 16%. In another embodiment,the fraction is 18%. In another embodiment, the fraction is 20%. Inanother embodiment, the fraction is 25%. In another embodiment, thefraction is 30%. In another embodiment, the fraction is 35%. In anotherembodiment, the fraction is 40%. In another embodiment, the fraction is45%. In another embodiment, the fraction is 50%. In another embodiment,the fraction is 60%. In another embodiment, the fraction is 70%. Inanother embodiment, the fraction is 80%. In another embodiment, thefraction is 90%. In another embodiment, the fraction is 100%.

In another embodiment, the fraction of the given uridine nucleotide isless than 8%. In another embodiment, the fraction is less than 10%. Inanother embodiment, the fraction is less than 5%. In another embodiment,the fraction is less than 3%. In another embodiment, the fraction isless than 1%. In another embodiment, the fraction is less than 2%. Inanother embodiment, the fraction is less than 4%. In another embodiment,the fraction is less than 6%. In another embodiment, the fraction isless than 12%. In another embodiment, the fraction is less than 15%. Inanother embodiment, the fraction is less than 20%. In anotherembodiment, the fraction is less than 30%. In another embodiment, thefraction is less than 40%. In another embodiment, the fraction is lessthan 50%. In another embodiment, the fraction is less than 60%. Inanother embodiment, the fraction is less than 70%.

In another embodiment, the terms “ribonucleotide,”“oligoribonucleotide,” and polyribonucleotide refers to, in oneembodiment, compounds comprising nucleotides in which the sugar moietyis ribose. In another embodiment, the term includes both RNA and RNAderivates in which the backbone is modified. Numerous RNA backbonemodifications are known in the art and contemplated in the presentinvention. In one embodiment, modified RNA is a PNA (peptide nucleicacid). PNA contain peptide backbones and nucleotide bases and are ableto bind, in another embodiment, to both DNA and RNA molecules. Inanother embodiment, the nucleotide is modified by replacement of one ormore phosphodiester bonds with a phosphorothioate bond. In anotherembodiment, the artificial nucleic acid contains any other variant ofthe phosphate backbone of native nucleic acids known in the art. Eachnucleic acid derivative represents a separate embodiment of the presentinvention.

Methods for production of nucleic acids having modified backbones arewell known in the art, and are described, for example in U.S. Pat. Nos.5,723,335 and 5,663,153 issued to Hutcherson et al. and related PCTpublication WO95/26204. Each method represents a separate embodiment ofthe present invention.

The nucleic acid of interest can be purified by any method known in theart, or any method to be developed, so long as the method ofpurification removes contaminants from the nucleic acid preparation andthereby substantially reduces the immunogenicity potential of thenucleic acid preparation. In one embodiment, the nucleic acid ofinterest is purified using high-performance liquid chromatography(HPLC). In another embodiment, the nucleic acid of interest is purifiedby contacting the nucleic acid of interest with the bacterial enzymeRNase III. In other various embodiments, any method of nucleic acidpurification that substantially reduces the immunogenicity of thenucleic acid preparation can be used. Non-limiting examples ofpurification methods that can be used with the compositions and methodsof the invention liquid chromatography separation and enzyme digestion,each used alone or in any combination, simultaneously or in any order.Non-limiting examples of liquid chromatography separation include HPLCand fast protein liquid chromatography (FPLC). Materials useful in theHPLC and FPLC methods of the invention include, but are not limited to,cross-linked poly styrene/divinylbenzene (PS/DVB), PS/DVB-C18,PS/DVB-alkylated, Helix DNA columns (Varian), Eclipse dsDNA AnalysisColumns (Agilent Technologies), Reverse-phase 5 (RPC-5) exchangematerial, DNAPac, ProSwift, and bio-inert UltiMate® 3000 Titaniumcolumns (Dionex). Enzymes useful in the enzyme digestion methods of theinvention include any enzyme able to digest any contaminant in a nucleicacid preparation of the invention, such as, for example a dsRNAcontaminant, and include but are not limited to, RNase III, RNase V1,Dicer, and Chipper (see Fruscoloni et al., 2002, PNAS 100:1639)Non-limiting examples of assays for assessing the purity of the nucleicacid of interest include a dot-blot assay, a Northern blot assay, and adendritic cell activation assay, as described elsewhere herein.

In another embodiment, the modified mRNA of the methods and compositionsof the present invention is significantly less immunogenic than anunmodified in vitro-synthesized mRNA molecule with the same sequence. Inanother embodiment, the modified mRNA molecule is 2-fold lessimmunogenic than its unmodified counterpart. In another embodiment,immunogenicity is reduced by a 3-fold factor. In another embodiment,immunogenicity is reduced by a 5-fold factor. In another embodiment,immunogenicity is reduced by a 7-fold factor. In another embodiment,immunogenicity is reduced by a 10-fold factor. In another embodiment,immunogenicity is reduced by a 15-fold factor. In another embodiment,immunogenicity is reduced by a fold factor. In another embodiment,immunogenicity is reduced by a 50-fold factor. In another embodiment,immunogenicity is reduced by a 100-fold factor. In another embodiment,immunogenicity is reduced by a 200-fold factor. In another embodiment,immunogenicity is reduced by a 500-fold factor. In another embodiment,immunogenicity is reduced by a 1000-fold factor. In another embodiment,immunogenicity is reduced by a 2000-fold factor. In another embodiment,immunogenicity is reduced by another fold difference.

In another embodiment, “significantly less immunogenic” refers to adetectable decrease in immunogenicity. In another embodiment, the termrefers to a fold decrease in immunogenicity (e.g. 1 of the folddecreases enumerated above). In another embodiment, the term refers to adecrease such that an effective amount of the modified mRNA can beadministered without triggering a detectable immune response. In anotherembodiment, the term refers to a decrease such that the modified mRNAcan be repeatedly administered without eliciting an immune responsesufficient to detectably reduce expression of the recombinant protein.In another embodiment, the decrease is such that the modified mRNA canbe repeatedly administered without eliciting an immune responsesufficient to eliminate detectable expression of the recombinantprotein.

Methods of determining immunogenicity are well known in the art, anddescribed in detail in U.S. Pat. No. 8,278,036 which is herebyincorporated by reference herein.

In another embodiment, the modified mRNA of the methods and compositionsof the present invention is translated in the cell more efficiently thanan unmodified mRNA molecule with the same sequence. In anotherembodiment, the modified mRNA exhibits enhanced ability to be translatedby a target cell. In another embodiment, translation is enhanced by afactor of 2-fold relative to its unmodified counterpart. In anotherembodiment, translation is enhanced by a 3-fold factor. In anotherembodiment, translation is enhanced by a 5-fold factor. In anotherembodiment, translation is enhanced by a 7-fold factor. In anotherembodiment, translation is enhanced by a 10-fold factor. In anotherembodiment, translation is enhanced by a 15-fold factor. In anotherembodiment, translation is enhanced by a 20-fold factor. In anotherembodiment, translation is enhanced by a 50-fold factor. In anotherembodiment, translation is enhanced by a 100-fold factor. In anotherembodiment, translation is enhanced by a 200-fold factor. In anotherembodiment, translation is enhanced by a 500-fold factor. In anotherembodiment, translation is enhanced by a 1000-fold factor. In anotherembodiment, translation is enhanced by a 2000-fold factor. In anotherembodiment, the factor is 10-1000-fold. In another embodiment, thefactor is 10-100-fold. In another embodiment, the factor is 10-200-fold.In another embodiment, the factor is 10-300-fold. In another embodiment,the factor is 10-500-fold. In another embodiment, the factor is20-1000-fold. In another embodiment, the factor is 30-1000-fold. Inanother embodiment, the factor is 50-1000-fold. In another embodiment,the factor is 100-1000-fold. In another embodiment, the factor is200-1000-fold. In another embodiment, translation is enhanced by anyother significant amount or range of amounts. Each possibilityrepresents a separate embodiment of the present invention.

Methods of determining translation efficiency are well known in the art,and include, e.g. measuring the activity of an encoded reporter protein(e.g luciferase or renilla or green fluorescent protein [Wall A A,Phillips A M et al, Effective translation of the second cistron in twoDrosophila dicistronic transcripts is determined by the absence ofin-frame AUG codons in the first cistron. J Biol Chem 2005; 280(30):27670-8]), or measuring radioactive label incorporated into thetranslated protein (Ngosuwan J, Wang N M et al, Roles of cytosolic Hsp70and Hsp40 molecular chaperones in post-translational translocation ofpre-secretory proteins into the endoplasmic reticulum. J Biol Chem 2003;278(9): 7034-42). Each method represents a separate embodiment of thepresent invention.

In another embodiment, the target cell of the method of the presentinvention is a dendritic cell. In another embodiment, the target cell ofthe method of the present invention is a macrophage. In anotherembodiment, the target cell of the method of the present invention is aB cell. In another embodiment, the target cell of the method of thepresent invention is another antigen presenting cell. In anotherembodiment, the target cell of methods of the present invention is amucosal cell. In another embodiment, the target cell of methods of thepresent invention is an epithelial cell. In another embodiment, the cellis a skin cell. In another embodiment, the cell is an epidermal cell. Inanother embodiment, the cell is a keratinocyte. In another embodiment,the cell is a Merkel cell, melanocyte or Langerhans cell. Eachpossibility represents a separate embodiment of the present invention.

Methods of Treatment and Uses of the Compositions

The present invention also provides methods of vaccinating a subjectagainst HSV and treating, impeding, inhibiting, reducing the incidenceof, or suppressing an HSV infection or a symptom or manifestationthereof, comprising administration of a composition of the presentinvention.

In one embodiment, the present invention provides a method for treatingan HSV infection in a subject, comprising contacting said subject with acomposition comprising one or more modified mRNAs, wherein each of saidmodified mRNAs encodes an HSV glycoprotein or immunogenic fragmentthereof.

In another embodiment, the present invention provides a method forsuppressing an HSV infection in a subject, comprising contacting saidsubject with a composition comprising one or more modified mRNAs,wherein each of said modified mRNAs encodes an HSV glycoprotein orimmunogenic fragment thereof.

In another embodiment, the present invention provides a method forinhibiting an HSV infection in a subject, comprising contacting saidsubject with a composition comprising one or more modified mRNAs,wherein each of said modified mRNAs encodes an HSV glycoprotein orimmunogenic fragment thereof.

In another embodiment, the present invention provides a method forreducing the incidence of HSV infection in a subject, comprisingcontacting said subject with a composition comprising one or moremodified mRNAs, wherein each of said modified mRNAs encodes an HSVglycoprotein or immunogenic fragment thereof.

In one embodiment, the HSV infection is an HSV-1 infection. In anotherembodiment, the HSV infection is an HSV-2 infection.

In one embodiment, the subject is administered HSV-1 glycoproteins formethods of treating, inhibiting, suppressing, etc. an HSV-1 infection.In another embodiment, the subject is administered HSV-2 glycoproteinsfor methods of treating, inhibiting, suppressing, etc. an HSV-2infection. In another embodiment, the subject is administered HSV-1glycoproteins for methods of treating, inhibiting, suppressing, etc. anHSV-1 infection, HSV-2 infection, or a combination thereof. In anotherembodiment, the subject is administered HSV-2 glycoproteins for methodsof treating, inhibiting, suppressing, etc. an HSV-1 infection, HSV-2infection, or a combination thereof. In one embodiment, administrationof HSV-1 glycoproteins (e.g., gC1, gD1, gE1, or a combination thereof)treats or prevents HSV-1 and HSV-2 infection. In another embodiment,administration of HSV-2 glycoproteins (e.g., gC2, gD2 and gE2, or acombination thereof) treats or prevents HSV-1 and HSV-2 infection.

According to this aspect and in one embodiment, the present inventionprovides a method for treating, suppressing, inhibiting, or reducing theincidence of Herpes Simplex Virus 1 (HSV-1) infection in a subject,comprising contacting said subject with a composition comprising one ormore modified mRNAs, wherein each of said modified mRNAs encodes anHSV-1 glycoprotein or immunogenic fragment thereof.

In one embodiment, the present invention provides a method for treating,suppressing, inhibiting, or reducing the incidence of Herpes SimplexVirus 2 (HSV-2) infection in a subject, comprising contacting saidsubject with a composition comprising one or more modified mRNAs,wherein each of said modified mRNAs encodes an HSV-2 glycoprotein orimmunogenic fragment thereof.

In one embodiment, said contacting is via administration to saidsubject.

In another embodiment, the present invention provides a method oftreating, suppressing, inhibiting, or reducing the incidence of an HSVinfection in a subject, the method comprising the step of administeringto said subject an immunogenic composition comprising modified mRNAsencoding: (a) an HSV gD or immunogenic fragment thereof; (b) an HSV gCor fragment thereof as described herein; (c) an HSV gE or fragmentthereof as described herein, or a combination thereof.

In another embodiment, the present invention provides a method oftreating, suppressing, inhibiting, or reducing the incidence of an HSVinfection in a subject, the method comprising the step of administeringto said subject an immunogenic composition comprising modified mRNAsencoding: (a) an HSV-2 gD or immunogenic fragment thereof; (b) an HSV-2gC or fragment thereof as described herein; and (c) an HSV-2 gE orfragment thereof as described herein, or a combination thereof.

In another embodiment, the present invention provides a method oftreating, suppressing, inhibiting, or reducing the incidence of an HSVinfection in a subject, the method comprising the step of administeringto said subject an immunogenic composition comprising modified mRNAsencoding: (a) an HSV-1 gD or immunogenic fragment thereof; (b) an HSV-1gC or fragment thereof as described herein; and (c) an HSV-1 gE orfragment thereof as described herein, or a combination thereof.

In another embodiment, the present invention provides a method ofinducing an anti-HSV immune response in a subject, the method comprisingthe step of administering to said subject an immunogenic compositioncomprising modified mRNAs encoding: (a) an HSV gD or immunogenicfragment thereof; (b) an HSV gC or fragment thereof as described herein;(c) an HSV gE or fragment thereof as described herein, or a combinationthereof.

In another embodiment, the present invention provides a method ofinducing an anti-HSV immune response in a subject, the method comprisingthe step of administering to said subject an immunogenic compositioncomprising modified mRNAs encoding: (a) an HSV-2 gD or immunogenicfragment thereof; (b) an HSV-2 gC or fragment thereof as describedherein; and (c) an HSV-2 gE or fragment thereof as described herein, ora combination thereof.

In another embodiment, the present invention provides a method ofinducing an anti-HSV immune response in a subject, the method comprisingthe step of administering to said subject an immunogenic compositioncomprising modified mRNAs encoding: (a) an HSV-1 gD or immunogenicfragment thereof; (b) an HSV-1 gC or fragment thereof as describedherein; and (c) an HSV-1 gE or fragment thereof as described herein, ora combination thereof.

In another embodiment, the present invention provides a method ofinhibiting a primary HSV infection in a subject, the method comprisingthe step of administering to the subject a composition of the presentinvention. In another embodiment, the present invention provides amethod of treating an HSV infection in a subject, the method comprisingthe step of administering to said subject a composition of the presentinvention. In another embodiment, the present invention provides amethod of reducing the incidence of an HSV infection in a subject, themethod comprising the step of administering to said subject acomposition of the present invention. In another embodiment, the presentinvention provides a method of inhibiting a flare following a primaryHSV infection in a subject, the method comprising the step ofadministering to said subject a composition of the present invention.

In one embodiment, the present invention provides methods of treatingand/or suppressing a primary HSV infection and/or a secondary HSVinfection. In one embodiment, a “primary” infection refers to afirst-time infection. In one embodiment, a “secondary” infection refersto a recurrence of an HSV infection.

In one embodiment, a “flare” or “recurrence” refers to reinfection ofskin tissue following latent neuronal HSV infection. In anotherembodiment, the terms refer to reactivation of HSV after a latencyperiod. In another embodiment, the terms refer to symptomatic HSVlesions following a non-symptomatic latency period.

In another embodiment, the present invention provides a method ofinhibiting spread of HSV. In one embodiment, the spread from DRG to skinis inhibited. In one embodiment, cell-to-cell spread of HSV isinhibited. In one embodiment, anterograde spread is inhibited. In oneembodiment, retrograde spread is inhibited. “DRG” refers, in oneembodiment, to a neuronal cell body and in another embodiment, containthe neuron cell bodies of nerve fibers. In another embodiment, the termrefers to any other definition of “DRG” used in the art. In anotherembodiment, spread of HSV to neural tissue is inhibited.

In another embodiment, the present invention provides a method ofinhibiting a recurrence following a primary HSV infection in a subject,the method comprising the step of administering to said subject acomposition of the present invention. In another embodiment, the presentinvention provides a method of preventing a recurrence following aprimary HSV infection in a subject, the method comprising the step ofadministering to said subject a composition of the present invention.

In another embodiment, the present invention provides a method ofinhibiting an HSV labialis following a primary HSV infection in asubject, the method comprising the step of administering to said subjecta composition of the present invention.

In another embodiment, the present invention provides a method ofpreventing a recurrence of an HSV infection, the method comprising thestep of administering to said subject a composition of the presentinvention. In another embodiment, the present invention provides amethod of diminishing the severity of a recurrence of an HSV infection,the method comprising the step of administering to said subject acomposition of the present invention. In another embodiment, the presentinvention provides a method of reducing the frequency of a recurrence ofan HSV infection, the method comprising the step of administering tosaid subject a composition of the present invention. In one embodiment,the present invention provides any of the described methods in anHIV-infected subject.

In another embodiment, the present invention provides a method oftreating an HSV encephalitis in a subject, the method comprising thestep of administering to said subject a composition of the presentinvention. In another embodiment, the present invention provides amethod of reducing the incidence of an HSV encephalitis in a subject,the method comprising the step of administering to said subject acomposition of the present invention. “HSV encephalitis” refers, in oneembodiment, to an encephalitis caused by a Herpes Simplex Virus-1 (HSV).In another embodiment, the term refers to an encephalitis associatedwith HSV. In another embodiment, the term refers to any other type ofHSV-mediated encephalitis known in the art.

In another embodiment, the present invention provides a method oftreating or reducing an HSV neonatal infection in a subject, the methodcomprising the step of administering to said subject a composition ofthe present invention.

In another embodiment, the present invention provides a method forintroducing an HSV glycoprotein to a cell of a subject, comprisingcontacting said cell with an in vitro-transcribed mRNA molecule encodingthe recombinant protein, wherein said in vitro-transcribed mRNA moleculefurther comprises a modified nucleoside, thereby introducing said HSVglycoprotein into said cell of said subject.

In another embodiment, the present invention provides a method forinducing a mammalian cell to produce an HSV glycoprotein, comprisingcontacting said mammalian cell with an in vitro-synthesized mRNAmolecule encoding the HSV glycoprotein, the in vitro-synthesized mRNAmolecule comprising a pseudouridine, thereby inducing said mammaliancell to produce said HSV glycoprotein.

It is to be understood that reference to HSV herein refers in oneembodiment, to HSV-1, while in another embodiment, to HSV-2, while inanother embodiment, to HSV-1 and HSV-2.

“HSV-1” refers, in another embodiment, to a Herpes Simplex Virus-1. Inanother embodiment, the term refers to a KOS strain. In anotherembodiment, the term refers to an F strain. In another embodiment, theterm refers to an NS strain. In another embodiment, the term refers to aCL101 strain. In another embodiment, the term refers to a “17” strain.In another embodiment, the term refers to a “17+syn” strain. In anotherembodiment, the term refers to a MacIntyre strain. In anotherembodiment, the term refers to an MP strain. In another embodiment, theterm refers to an HF strain. In another embodiment, the term refers toany other HSV-1 strain known in the art.

“HSV-2” refers, in another embodiment, to a Herpes Simplex Virus-2. Inanother embodiment, the term refers to an HSV-2 333 strain. In anotherembodiment, the term refers to a 2.12 strain. In another embodiment, theterm refers to an HG52 strain. In another embodiment, the term refers toan MS strain. In another embodiment, the term refers to a G strain. Inanother embodiment, the term refers to a 186 strain. In anotherembodiment, the term refers to any other HSV-2 strain known in the art.

In another embodiment, the present invention provides a method ofvaccinating a subject against an HSV infection, the method comprisingthe step of administering to said subject a composition of the presentinvention. In another embodiment, the present invention provides amethod of suppressing an HSV infection in a subject, the methodcomprising the step of administering to said subject a composition ofthe present invention. In another embodiment, the present inventionprovides a method of impeding an HSV infection in a subject, the methodcomprising the step of administering to said subject a composition ofthe present invention. In another embodiment, the present inventionprovides a method of impeding a primary HSV infection in a subject, themethod comprising the step of administering to said subject acomposition of the present invention. In another embodiment, the presentinvention provides a method of impeding neuronal HSV spread in asubject, the method comprising the step of administering to said subjecta composition of the present invention.

The terms “impeding an HSV infection” and “impeding a primary HSVinfection” refer, in another embodiment, to decreasing the titer ofinfectious virus. In another embodiment, the terms refer to decreasingthe extent of viral replication.

In another embodiment, the present invention provides a method ofreducing the incidence of an HSV-mediated herpetic ocular disease in asubject, the method comprising the step of administering to said subjecta composition of the present invention. In another embodiment, thepresent invention provides a method of treating an HSV-1 cornealinfection or herpes keratitis in a subject, the method comprising thestep of administering to said subject a composition of the presentinvention. In another embodiment, the present invention provides amethod of reducing the incidence of an HSV-1 corneal infection or herpeskeratitis in a subject, the method comprising the step of administeringto said subject a composition of the present invention.

In another embodiment, the present invention provides a method oftreating, suppressing or inhibiting an HSV genital infection, the methodcomprising the step of administering to said subject a composition ofthe present invention. In another embodiment, the present inventionprovides a method of treating, suppressing or inhibiting anymanifestation of recurrent HSV infection, the method comprising the stepof administering to said subject a composition of the present invention.

In another embodiment, the present invention provides a method ofreducing the incidence of an HSV-mediated genital ulcer disease in asubject, the method comprising the step of administering to said subjecta composition of the present invention. In another embodiment, thepresent invention provides a method of impeding an establishment of alatent HSV infection in a subject, the method comprising the step ofadministering to said subject a composition of the present invention.

In one embodiment, the present invention provides a method of treating,suppressing or inhibiting a genital herpes infection in a subject,comprising the step of administering to said subject a composition ofthe present invention. In another embodiment, the present inventionprovides a method of treating, suppressing or inhibiting an oral herpesinfection in a subject, comprising the step of administering to saidsubject a composition of the present invention.

In another embodiment, the present invention provides a method ofreducing the incidence of an HSV-mediated encephalitis in a subject, themethod comprising the step of administering to said subject acomposition of the present invention.

In another embodiment, the herpes-mediated encephalitis treated orprevented by a method of the present invention is a focal herpesencephalitis. In another embodiment, the herpes-mediated encephalitis isa neonatal herpes encephalitis. In another embodiment, theherpes-mediated encephalitis is any other type of herpes-mediatedencephalitis known in the art.

In another embodiment, the present invention provides a method oftreating or reducing the incidence of a disease, disorder, or symptomassociated with or secondary to an HSV-mediated encephalitis in asubject, the method comprising the step of administering to said subjecta composition of the present invention.

In another embodiment, the present invention provides a method oftreating, reducing the pathogenesis of, ameliorating the symptoms of,ameliorating the secondary symptoms of, reducing the incidence of,prolonging the latency to a relapse of an HSV infection in a subject,comprising the step of administering to the subject a composition of thepresent invention.

In another embodiment, the present invention provides a method ofprotecting a subject against formation of a zosteriform lesion or ananalogous outbreak in a human subject. In another embodiment, thepresent invention provides a method of inhibiting the formation of anHSV zosteriform lesion or an analogous outbreak in a human subject.

“Zosteriform” refers, in one embodiment, to skin lesions characteristicof an HSV infection, particularly during reactivation infection, which,in one embodiment, begin as a rash and follow a distribution neardermatomes, commonly occurring in a strip or belt-like pattern. In oneembodiment, the rash evolves into vesicles or small blisters filled withserous fluid. In one embodiment, zosteriform lesions form in mice as aresult of contact with HSV. In another embodiment, zosteriform lesionsform in humans as a result of contact with HSV. “Zosteriform spread”refers, in one embodiment, to an HSV infection that spreads from theganglia to secondary skin sites within the dermatome. In anotherembodiment, the term refers to spread within the same dermatome as theinitial site of infection. In another embodiment, the term refers to anyother definition of “zosteriform spread” known in the art. “Outbreak”,in another embodiment, refers to a sudden increase in symptoms of adisease or in the spread or prevalence of a disease, and in oneembodiment, refers to a sudden increase in zosteriform lesions, while inanother embodiment, “outbreak” refers to a sudden eruption ofzosteriform lesions.

In one embodiment, the present invention provides a method of impedingthe formation of a dermatome lesion or an analogous condition in asubject. In one embodiment, dermatome lesions form as a result ofcontact with HSV. In another embodiment, dermatome lesions most oftendevelop when the virus reactivates from latency in the ganglia and inone embodiment, spreads down nerves, in one embodiment, causing arecurrent infection.

It is to be understood that the methods of the present invention may beused to treat, inhibit, suppress, etc an HSV infection or primary orsecondary symptoms related to such an infection following exposure ofthe subject to HSV. In another embodiment, the subject has been infectedwith HSV before vaccination. In another embodiment, the subject is atrisk for HSV infection. In another embodiment, whether or not thesubject has been infected with HSV at the time of vaccination,vaccination by a method of the present invention is efficacious intreating, inhibiting, suppressing, etc. an HSV infection or primary orsecondary symptoms related to such an infection.

In one embodiment, “treating” refers to either therapeutic treatment orprophylactic or preventative measures, wherein the object is to preventor lessen the targeted pathologic condition or disorder as describedhereinabove. Thus, in one embodiment, treating may include directlyaffecting or curing, suppressing, inhibiting, preventing, reducing theseverity of, delaying the onset of, reducing symptoms associated withthe disease, disorder or condition, or a combination thereof. Thus, inone embodiment, “treating” refers inter alia to delaying progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In one embodiment, “preventing”refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

In one embodiment, the compositions and methods of the present inventionare effective in lowering HSV acquisition rates, duration of HSVinfection, frequency of HSV reactivation, or a combination thereof. Inanother embodiment, the compositions and methods of the presentinvention are effective in treating or inhibiting genital ulcer disease,which in one embodiment, entails decreasing the severity or frequency ofHSV genital ulcer disease. In one embodiment, the compositions andmethods of the present invention block immune evasion from complement.In one embodiment, vaccination with mRNA-encoded HSV subunits mayproduce high titers of neutralizing antibodies or potent T-cellresponses; however, upon subsequent infection, HSV immune evasionmolecules may block the activities of antibodies or T cells, therebyreducing composition efficacy. In one embodiment, the compositions andmethods of the present invention incorporate strategies to block virusmediated immune evasion by, in one embodiment, enhancing theeffectiveness of e.g. a gD-1 subunit composition using gC-1 to preventimmune evasion from complement.

In one embodiment, studies in guinea pigs and mice suggest that viralload in ganglia correlates with the frequency of recurrent HSVinfections. Thus, in one embodiment, the compositions and methods of thepresent invention are useful for preventing or inhibiting recurrent HSVinfections. In one embodiment, antibodies to e.g. gC-1 block domainsinvolved in immune evasion, which enhances complement activity, improvesneutralizing activity of anti-gD-1 IgG, increases antibody- andcomplement-dependent cellular cytotoxicity, and augmentscomplement-mediated neutralization and lysis of infected cells.

In one embodiment, symptoms are primary, while in another embodiment,symptoms are secondary. In one embodiment, “primary” refers to a symptomthat is a direct result of the subject viral infection, while in oneembodiment, “secondary” refers to a symptom that is derived from orconsequent to a primary cause. In one embodiment, the compositions andstrains for use in the present invention treat primary or secondarysymptoms or secondary complications related to HSV infection.

In another embodiment, “symptoms” may be any manifestation of an HSVinfection, comprising blisters, ulcerations, or lesions on the urethra,cervix, upper thigh, and/or anus in women and on the penis, urethra,scrotum, upper thigh, and anus in men, inflammation, swelling, fever,flu-like symptoms, sore mouth, sore throat, pharyngitis, pain, blisterson tongue, mouth or lips, ulcers, cold sores, neck pain, enlarged lymphnodes, reddening, bleeding, itching, dysuria, headache, muscle pain,etc., or a combination thereof.

In another embodiment, the disease, disorder, or symptom is fever. Inanother embodiment, the disease, disorder, or symptom is headache. Inanother embodiment, the disease, disorder, or symptom is stiff neck. Inanother embodiment, the disease, disorder, or symptom is seizures. Inanother embodiment, the disease, disorder, or symptom is partialparalysis. In another embodiment, the disease, disorder, or symptom isstupor. In another embodiment, the disease, disorder, or symptom iscoma. In another embodiment, the disease, disorder, or symptom is anyother disease, disorder, or symptom known in the art that is associatedwith or secondary to a herpes-mediated encephalitis.

Methods of determining the presence and severity of herpes-mediatedencephalitis are well known in the art, and are described, for example,in Bonkowsky J L et al. (Herpes simplex virus central nervous systemrelapse during treatment of infantile spasms with corticotropin.Pediatrics. 2006 May; 117(5):e1045-8) and Khan O A, et al. (Herpesencephalitis presenting as mild aphasia: case report. BMC Fam Pract.2006 Mar. 24; 7:22). Each method represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method oftreating or reducing the incidence of a disease, disorder, or symptomassociated with an HSV infection in a subject, the method comprising thestep of administering to said subject a composition of the presentinvention.

In another embodiment, the disease, disorder, or symptom secondary to anHSV infection is oral lesions. In another embodiment, the disease,disorder, or symptom is genital lesions. In another embodiment, thedisease, disorder, or symptom is oral ulcers. In another embodiment, thedisease, disorder, or symptom is genital ulcers. In another embodiment,the disease, disorder, or symptom is fever. In another embodiment, thedisease, disorder, or symptom is headache. In another embodiment, thedisease, disorder, or symptom is muscle ache. In another embodiment, thedisease, disorder, or symptom is swollen glands in the groin area. Inanother embodiment, the disease, disorder, or symptom is painfulurination. In another embodiment, the disease, disorder, or symptom isvaginal discharge. In another embodiment, the disease, disorder, orsymptom is blistering. In another embodiment, the disease, disorder, orsymptom is flu-like malaise. In another embodiment, the disease,disorder, or symptom is keratitis. In another embodiment, the disease,disorder, or symptom is herpetic whitlow. In another embodiment, thedisease, disorder, or symptom is Bell's palsy. In another embodiment,the disease, disorder, or symptom is herpetic erythema multiforme. Inanother embodiment, the disease, disorder, or symptom is a lower backsymptom (e.g. numbness, tingling of the buttocks or the area around theanus, urinary retention, constipation, and impotence). In anotherembodiment, the disease, disorder, or symptom is a localized eczemaherpeticum. In another embodiment, the disease, disorder, or symptom isa disseminated eczema herpeticum. In another embodiment, the disease,disorder, or symptom is a herpes gladiatorum. In another embodiment, thedisease, disorder, or symptom is a herpetic sycosis. In anotherembodiment, the disease, disorder, or symptom is an esophageal symptom(e.g. difficulty swallowing or burning, squeezing throat pain whileswallowing, weight loss, pain in or behind the upper chest whileswallowing). In another embodiment, the disease, disorder, or symptom isany other disease, disorder, or symptom that is known in the art. Eachdisease, disorder, and symptom represents a separate embodiment of thepresent invention.

Thus, in one embodiment, the compositions and methods of the instantinvention treat, suppress, inhibit, or reduce the incidence of theinfection itself, while in another embodiment, the compositions andmethods of the instant invention treat, suppress, inhibit, or reduce theincidence of primary symptoms of the infection, while in anotherembodiment, the compositions and methods of the instant invention treat,suppress, inhibit, or reduce the incidence of secondary symptoms of theinfection. It is to be understood that the compositions and methods ofthe instant invention may affect any combination of the infection, theprimary symptoms caused by the infection, and secondary symptoms relatedto the infection.

The HSV infection that is treated or ameliorated by methods andcompositions of the present invention is, in another embodiment, agenital HSV infection. In another embodiment, the HSV infection is anoral HSV infection. In another embodiment, the HSV infection is anocular HSV infection. In another embodiment, the HSV infection is adermatologic HSV infection.

In another embodiment, the present invention provides a method ofreducing the incidence of a disseminated HSV infection in a subject, themethod comprising the step of administering to said subject acomposition of the present invention.

In another embodiment, the present invention provides a method ofreducing the incidence of a neonatal HSV infection in an offspring of asubject, the method comprising the step of administering to said subjecta composition of the present invention.

In another embodiment, the present invention provides a method ofreducing a transmission of an HSV infection from a subject to anoffspring thereof, the method comprising the step of administering tosaid subject a composition of the present invention.

In another embodiment, the offspring is an infant. In anotherembodiment, the transmission that is reduced or inhibited istransmission during birth. In another embodiment, transmission duringbreastfeeding is reduced or inhibited. In another embodiment, thetransmission that is reduced or inhibited is any other type ofparent-to-offspring transmission known in the art.

In another embodiment, the present invention provides a method ofreducing a severity of a neonatal HSV infection in an offspring of asubject, the method comprising the step of administering to said subjecta composition of the present invention.

In one embodiment, the present invention provides a method of treating,suppressing, inhibiting, or reducing the incidence of an HSV infectionin a subject infected with HIV, the method comprising the step ofadministering to said subject a composition comprising: (a) a modifiedmRNA encoding HSV gC protein or fragment thereof; (b) a modified mRNAencoding HSV gE protein or fragment thereof; and (c) an adjuvant. Inanother embodiment, the present invention provides a method of treating,suppressing, inhibiting, or reducing the incidence of an HSV infectionin a subject infected with HIV, the method comprising the step ofadministering to said subject a composition comprising: (a) a modifiedmRNA encoding HSV gC protein or fragment thereof, wherein said fragmentcomprises either a C3b-binding domain thereof, a properdin interferingdomain thereof, a C5 interfering domain thereof, or a fragment of saidC3b-binding domain, properdin interfering domain, or C5-interferingdomain; (b) a modified mRNA encoding HSV gE protein or fragment thereof,wherein said fragment comprises AA 24-409 or a fragment thereof; and (c)an adjuvant.

In another embodiment, the present invention provides a method oftreating an HSV infection in a subject infected with HIV, the methodcomprising the step of administering to said subject a composition ofthe present invention. In another embodiment, the present inventionprovides a method of suppressing an HSV infection in a subject infectedwith HIV, the method comprising the step of administering to saidsubject a composition of the present invention. In another embodiment,the present invention provides a method of inhibiting an HSV infectionin a subject infected with HIV, the method comprising the step ofadministering to said subject a composition of the present invention. Inanother embodiment, the present invention provides a method of reducingthe incidence of an HSV infection in a subject infected with HIV, themethod comprising the step of administering to said subject acomposition of the present invention. In another embodiment, the presentinvention provides a method of preventing an HIV infection, the methodcomprising the step of administering to said subject an HSV compositionof the present invention. In one embodiment, HSV infection increases therisk of HIV infection, and protection against HSV infection decreasesthe risk of HIV infection. Thus, in one embodiment, the presentinvention provides a method of decreasing the risk of an HIV infection,the method comprising the step of administering to said subject acomposition of the present invention.

In one embodiment, the composition for use in the methods of the presentinvention elicits an immune response against HSV. In another embodiment,the composition for use in the methods of the present invention elicitsan immune response against HSV-1. In another embodiment, the compositionfor use in the methods of the present invention elicits an immuneresponse against HSV-2. In another embodiment, the composition comprisesmodified mRNAs encoding gD and gC proteins. In another embodiment, thecomposition comprises modified mRNAs encoding gE and gD proteins. Inanother embodiment, the composition comprises modified mRNAs encoding gCand gE proteins. In another embodiment, the composition comprisesmodified mRNAs encoding gE, gD, and gC proteins. In another embodiment,the composition comprises modified mRNAs encoding gE, gD, or gC protein.In another embodiment, the proteins encoded by the modified mRNAs areHSV-1 proteins. In another embodiment, the proteins encoded by themodified mRNAs are HSV-2 proteins. In another embodiment, the proteinsencoded by the modified mRNAs comprise both HSV-1 and HSV-2 proteins.

It is to be understood that, in one embodiment, a subject according toany of the embodiments described herein may be a subject infected with,or in another embodiment, susceptible to infection with HSV. In oneembodiment, a subject may be infected with, or in another embodiment,susceptible to infection with at least one other pathogen. In oneembodiment, a subject may be immunocompromised. In one embodiment, thesubject is infected by HSV, while in another embodiment, the subject isat risk for infection by HSV, which in one embodiment, is a subject whois a neonate, in another embodiment, immunocompromised, in anotherembodiment, elderly, and in another embodiment, an immunocompromisedneonate or an immunocompromised elderly subject.

In another embodiment, the compositions of the present invention andtheir related uses may suppress, inhibit, prevent or treat an HIVinfection in a subject. In one embodiment, the compositions of thepresent invention and their related uses may treat secondarycomplications of HIV infection, which in one embodiment, areopportunistic infections, neoplasms, neurologic abnormalities, orprogressive immunologic deterioration. In another embodiment, themethods comprise treating acquired immunodeficiency syndrome (AIDS). Inanother embodiment, the methods comprise treating a decline in thenumber of CD4⁺ T lymphocytes.

In another embodiment, the present invention provides a method ofreducing HIV-1 transmission to an offspring, the method comprising thestep of administering to a subject a composition of the presentinvention. As is known in the art, HSV-2 infection increases HIV-1 viralshedding in genital secretions (Nagot N et al., Reduction of HIV-1 RNAlevels with therapy to suppress herpes simplex virus. N Engl J Med. 2007Feb. 22; 356(8):790-9). Thus, methods of the present invention ofinhibiting HSV-2 infection are also efficacious for reducing HIV-1transmission to an offspring. In another embodiment, the mutant HSVstrain is an HSV-1 strain. In another embodiment, the mutant HSV strainis an HSV-2 strain.

In another embodiment, the present invention provides a method ofreducing HIV-1 transmission to a sexual partner, the method comprisingthe step of administering to a subject a composition of the presentinvention. As is known in the art, HSV-2 infection increases HIV-1 viralshedding in genital secretions. Thus, methods of the present inventionof inhibiting HSV-2 infection are also efficacious for reducing HIV-1transmission to a sexual partner. In another embodiment, the mutant HSVstrain is an HSV-1 strain. In another embodiment, the mutant HSV strainis an HSV-2 strain.

In another embodiment, the present invention provides a method ofreducing susceptibility to HIV-1, the method comprising the step ofadministering to a subject a composition of the present invention. As isknown in the art, HSV-2 infection increases HIV-1 replication (OuedraogoA et al., Impact of suppressive herpes therapy on genital HIV-1 RNAamong women taking antiretroviral therapy: a randomized controlledtrial. AIDS. 2006 Nov. 28; 20(18):2305-13). Thus, methods of the presentinvention of inhibiting HSV-2 infection are also efficacious forreducing susceptibility to HIV-1. In another embodiment, the mutant HSVstrain is an HSV-1 strain. In another embodiment, the mutant HSV strainis an HSV-2 strain.

Thus, in one embodiment, the present invention provides a method ofinhibiting a primary HSV infection in an HIV-infected subject,comprising the step of administering to said subject a composition ofthe present invention. In another embodiment, the present inventionprovides a method of treating or reducing the incidence of an HSVinfection in an HIV-infected subject, comprising the step ofadministering to said subject a composition of the present invention. Inanother embodiment, the present invention provides a method ofinhibiting a flare, recurrence, or HSV labialis following a primary HSVinfection in an HIV-infected subject, the method comprising the step ofadministering to said subject a composition of the present invention. Inone embodiment, administration of a composition of the present inventionan anti-HSV immune response.

In another embodiment, the present invention provides a method forinducing an immune response in a subject, the method comprising the stepof administering to said subject a nucleoside modified mRNA compositionof the present invention. In another embodiment, the immune responsecomprises a CD4 immune response. In another embodiment, the immuneresponse comprises a CD8 immune response. In another embodiment, theimmune response comprises a T follicular helper cell immune response. Inanother embodiment, the immune response comprises a germinal center Bcell immune response. In another embodiment, the immune responsecomprises an IgG antibody response to gC2, gD2, gE2 or a combinationthereof.

In another embodiment, the present invention provides a method oftreating a Herpes Simplex Virus (HSV) infection in a subject, the methodcomprising the step of intramuscularly administering to said subject anucleoside modified mRNA composition of the present invention. Inanother embodiment, the invention provides a method of suppressing,inhibiting, or reducing the incidence of a Herpes Simplex Virus (HSV)infection in a subject, the method comprising the step ofintramuscularly administering to said subject a nucleoside modified mRNAcomposition of the present invention.

Administration and Pharmaceutical Regimens

Compositions of the present invention can be, in another embodiment,administered to a subject by any method known to a person skilled in theart, such as parenterally, paracancerally, transmucosally,transdermally, intramuscularly, intravenously, intra-dermally,subcutaneously, intra-peritonealy, intra-ventricularly, intra-cranially,intra-vaginally, intra-nasally, intra-tumorally, or topically.

“Administering,” in another embodiment, refers to directly introducinginto a subject by injection or other means a composition of the presentinvention. In another embodiment, “administering” refers to contacting acell of the subject's immune system with a composition or modified mRNAencoding HSV protein or mixture thereof.

In another embodiment of the methods and compositions of the presentinvention, the compositions are administered orally, and are thusformulated in a form suitable for oral administration, i.e. as a solidor a liquid preparation. Suitable solid oral formulations includetablets, capsules, pills, granules, pellets and the like. Suitableliquid oral formulations include solutions, suspensions, dispersions,emulsions, oils and the like. In another embodiment of the presentinvention, the active ingredient is formulated in a capsule. Inaccordance with this embodiment, the compositions of the presentinvention comprise, in addition to the active compound and the inertcarrier or diluent, a hard gelating capsule.

In other embodiments, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intramuscular injection of a liquidpreparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In anotherembodiment, the pharmaceutical compositions are administeredintravenously and are thus formulated in a form suitable for intravenousadministration. In another embodiment, the pharmaceutical compositionsare administered intra-arterially and are thus formulated in a formsuitable for intra-arterial administration. In another embodiment, thepharmaceutical compositions are administered intramuscularly and arethus formulated in a form suitable for intramuscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the compositions or their physiologically toleratedderivatives are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the composition is administered as a suppository,for example a rectal suppository or a urethral suppository. In anotherembodiment, the pharmaceutical composition is administered bysubcutaneous implantation of a pellet. In another embodiment, the pelletprovides for controlled release of agent over a period of time.

In a preferred embodiment, pharmaceutical compositions are administeredintramuscularly, subcutaneously or intradermally.

“Effective dosage” of the modified mRNA, refers, in another embodiment,to an amount sufficient to exert a therapeutic effect. In anotherembodiment, the term refers to an amount sufficient to elicit expressionof a detectable amount of the encoded protein. Each possibilityrepresents a separate embodiment of the present invention.

Methods for measuring the dose of a modified mRNA encoding an HSVglycoprotein (e.g. in human subjects) are well known in the art, andinclude, for example, dose-escalating trials. Each method represents aseparate embodiment of the present invention.

In some embodiments, any of the HSV compositions of and for use in themethods of this invention will comprise a modified mRNA encoding HSVprotein or combination of modified mRNAs encoding HSV proteins of thepresent invention, in any form or embodiment as described herein. Insome embodiments, any of the compositions of and for use in the methodswill consist of a modified mRNA encoding HSV protein or combination ofmodified mRNA encodings HSV proteins of the present invention, in anyform or embodiment as described herein. In some embodiments, thecompositions of this invention will consist essentially of a modifiedmRNA encoding an HSV protein or combination of modified mRNAs encodingHSV proteins of the present invention, in any form or embodiment asdescribed herein. In some embodiments, the term “comprise” refers to theinclusion of modified mRNA encoding other HSV proteins, as well asinclusion of modified mRNA encoding other proteins that may be known inthe art. In some embodiments, the term “consisting essentially of”refers to a composition, which has the modified mRNA encoding a specificHSV protein or fragment thereof. However, other components may beincluded that are not involved directly in the utility of the modifiedmRNA(s) encoding HSV protein(s). In some embodiments, the term“consisting” refers to a composition having a modified mRNA encodingparticular HSV protein or fragment or combination of modified mRNAsencoding HSV proteins or fragments of the present invention, in any formor embodiment as described herein.

In another embodiment, the present invention provides a composition fortreating HSV-1 or a symptom or manifestation thereof, the compositioncomprising a modified mRNA of the present invention.

In another embodiment, the present invention provides a composition fortreating HSV-2 or a symptom or manifestation thereof, the compositioncomprising a modified mRNA of the present invention.

It is to be understood that the compositions, and methods of the presentinvention may be used in non-HSV herpesvirus as well, which in oneembodiment, proteins gD, gE, or gC proteins that are, in one embodiment,70% homologous, in another embodiment, 80% homologous, in anotherembodiment, 85% homologous, in another embodiment, 90% homologous, inanother embodiment, 95% homologous, in another embodiment, 98%homologous, and in another embodiment, 100% homologous to the gD, gE, orgC proteins of HSV-1, or in another embodiment, of HSV-2. In oneembodiment, such compositions may be useful in suppressing, inhibiting,preventing, or treating, cancers, or in another embodiment, tumors. Inone embodiment, non-HSV herpesvirus comprise Varicella Zoster Virus(VZV), Epstein-Barr virus (EBV), EBNA, cytomegalovirus (CMV), and humanherpesvirus-6 (HHV-6).

In another embodiment, of methods of the present invention, acomposition of the present invention is administered once. In anotherembodiment, the composition is administered twice. In anotherembodiment, the composition is administered three times. In anotherembodiment, the composition is administered four times. In anotherembodiment, the composition is administered at least four times. Inanother embodiment, the composition is administered more than fourtimes.

In another embodiment, the dosage is a daily dose. In anotherembodiment, the dosage is a weekly dose. In another embodiment, thedosage is a monthly dose. In another embodiment, the dosage is an annualdose. In another embodiment, the dose is one is a series of a definednumber of doses. In another embodiment, the dose is a one-time dose.

In one embodiment, any of the booster doses described hereinabove isadministered following a priming dose comprising one or modified moremRNAs encoding HSV-1 proteins or immunogenic fragments thereof. Inanother embodiment, any of the booster doses described hereinabove isadministered following a priming vaccination comprising one or moremodified more mRNAs encoding HSV-2 proteins or immunogenic fragmentsthereof.

In one embodiment, a subject is immunized with a single administrationof the composition. In another embodiment, a subject is immunized with asingle dose. In another embodiment, a subject is immunized with twodoses. In another embodiment, a subject is immunized with three doses.In another embodiment, a subject is immunized with four doses. Inanother embodiment, a subject is immunized with five doses.

In one embodiment, all the components of the composition are provided inequal concentrations. According to this aspect and in one embodiment,modified mRNAs encoding gC, gD, and gE are provided in a ratio of 1:1:1.In another embodiment, modified mRNAs encoding gC, gD, and gE areprovided in a ratio of 5:2:5. In another embodiment, modified mRNAsencoding gC and gD are provided in a ratio of 1:1. In anotherembodiment, modified mRNAs encoding gC and gE are provided in a ratio of1:1. In another embodiment, modified mRNAs encoding gD and gE areprovided in a ratio of 1:1.

In one embodiment, modified mRNAs encoding gC, gD, gE, or a combinationthereof, or combined with other HSV glycoproteins, are administered in asingle composition at the same site and by the same route, while inanother embodiment, modified mRNAs encoding gC, gD, and gE areadministered in separate compositions at separate sites but by the sameroute of administration, or in another embodiment, modified mRNAsencoding gC, gD, and gE are administered in separate compositions atseparate sites and by different routes of administration, or in anotherembodiment, modified mRNAs encoding gC, gD, and gE are administered inseparate compositions at the same site and by different routes ofadministration (e.g. injection and topical).

In one embodiment, the methods of the present invention include aone-time or single administration of compositions comprising one or morenucleoside modified mRNAs of the present invention. In anotherembodiment, the methods of the present invention include administrationof compositions comprising one or more nucleoside modified mRNAs in aprime and boost approach. In one embodiment, the methods of the presentinvention further comprise the step of administering to said subject oneor more additional administrations of said nucleoside modified mRNAcomposition subsequent to the first administration.

In another embodiment, the methods of the present invention compriseadministering a composition comprising one or more nucleoside modifiedmRNAs encoding one or more HSV glycoproteins as a first administrationand a composition comprising one or more HSV glycoproteins as a secondor subsequent administration. In one embodiment, the HSV glycoproteinsencoded by the mRNA in the first (or prime) administration are the sameglycoproteins in the second or subsequent (or boost) administration. Inanother embodiment, a composition comprising one or more HSVglycoproteins is administered as a first administration, and acomposition comprising one or more nucleoside modified mRNAs encodingone or more HSV glycoproteins is administered as a second or subsequentadministration. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, modified mRNAs encoding gC, gD, and gE areadministered simultaneously followed by a booster dose of modified mRNAencoding gD without modified mRNAs encoding gC or gE. In anotherembodiment, modified mRNAs encoding gC, gD, and gE are administeredsimultaneously followed by a booster dose of modified mRNA encoding gCwithout modified mRNAs encoding gD or gE. In another embodiment,modified mRNAs encoding gC, gD, and gE are administered simultaneouslyfollowed by a booster dose of modified mRNA encoding gE without modifiedmRNAs encoding gD or gC. In another embodiment, modified mRNAs encodinggC, gD, and gE are administered simultaneously followed by a boosterdose of modified mRNAs encoding gC and gD without modified mRNAsencoding gE. In another embodiment, modified mRNAs encoding gC, gD, andgE are administered simultaneously followed by a booster dose ofmodified mRNAs encoding gC and gE without modified mRNAs encoding gD. Inanother embodiment, modified mRNAs encoding gC, gD, and gE areadministered simultaneously followed by a booster dose of modified mRNAsencoding gD and gE without modified mRNAs encoding gE. In one embodimentthe booster administration is performed at the same site and by the samemode of administration as the priming administration. In anotherembodiment, the booster administration is performed at a different sitefrom the priming administration but by the same mode of administrationas the priming administration. In one embodiment the boosteradministration is performed at the same site but by different mode ofadministration than priming administration. In another embodiment, thebooster administration is performed at a different site and by differentmode of administration than priming administration.

In one embodiment, the modified mRNA induces a detectably lower innateimmune response than the same quantity of unmodified RNA having the samesequence.

In one embodiment, the effectiveness of the compositions and methods ofthe present invention are dependent on the presence of complement, whilein another embodiment, the compositions and methods of the presentinvention are not dependent on the presence of complement. In oneembodiment, the effectiveness of some of the compositions for use in themethods of the present invention are dependent on the presence ofcomplement, while others are not. In one embodiment, the anti-gCantibody is dependent on complement for its effectiveness against HSV.

In one embodiment, complement is an important contributor to innate andacquired immunity. In one embodiment, complement activation facilitatesvirus neutralization by particle phagocytosis and lysis, functions as achemoattractant for neutrophils and macrophages, and enhances B and Tcell responses. In one embodiment, HSV-1 gC binds complement C3b andblocks C5 and properdin interaction with C3b, which inhibit complementactivation and complement-mediated virus neutralization. In oneembodiment, a gC-1 domain that interacts with complement is locatedwithin amino acids 33 to 133 and blocks C5 and properdin binding to C3b,and in one embodiment, a gC-1 domain that interacts with complementextends from amino acids 124 to 366 and directly binds C3b. In oneembodiment, an HSV-1 gC mutant virus deleted in the C3b binding domainis more susceptible to complement-mediated virus neutralization in vitroand less virulent than wild-type (WT) virus in the mouse flank model.Therefore, in one embodiment, the interaction between gC-1 and C3benhances HSV-1 virulence, and in one embodiment, blocking the gC-1domain is effective in preventing or treating HSV-1 infection.

In one embodiment, the compositions and methods of the present inventionare for use in human subjects, while in another embodiment, they are foruse in animal subjects. In another embodiment, the subject is mammalian.In another embodiment, the subject is any organism susceptible toinfection by HSV. In one embodiment, the subject is murine, bovine,ovine, canine, feline, equine, porcine, etc. In one embodiment, thecompositions and methods of the present invention are effective in malesubjects. In another embodiment, the compositions and methods of thepresent invention are effective in female subjects. In one embodiment,the compositions and methods of the present invention are effective inseronegative subjects. In another embodiment, the compositions andmethods of the present invention are effective in seropositive subjects.

Pharmaceutical Formulations

In one embodiment, a method of present invention further comprisesmixing the modified mRNA with a transfection reagent prior to the stepof contacting. In another embodiment, a method of present inventionfurther comprises administering the modified mRNA together with thetransfection reagent. In another embodiment, the transfection reagent isa cationic lipid reagent.

In another embodiment, the transfection reagent is a lipid-basedtransfection reagent. In another embodiment, the transfection reagent isa protein-based transfection reagent. In another embodiment, thetransfection reagent is a polyethyleneimine based transfection reagent.In another embodiment, the transfection reagent is calcium phosphate. Inanother embodiment, the transfection reagent is Lipofectin® orLipofectamine®. In another embodiment, the transfection reagent is anyother transfection reagent known in the art.

In another embodiment, the transfection reagent forms a liposome.Liposomes, in another embodiment, increase intracellular stability,increase uptake efficiency and improve biological activity.

In another embodiment, liposomes are hollow spherical vesicles composedof lipids arranged in a similar fashion as those lipids which make upthe cell membrane. They have, in another embodiment, an internal aqueousspace for entrapping water soluble compounds and range in size from 0.05to several microns in diameter. In another embodiment, liposomes candeliver RNA to cells in a biologically active form (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid).

Each type of transfection reagent represents a separate embodiment ofthe present invention.

In another embodiment, a modified mRNA of the present invention isencapsulated in a nanoparticle. Methods for nanoparticle packaging arewell known in the art, and are described, for example, in Bose S, et al(Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection ofHuman Lung Epithelial Cells. J. Virol. 78:8146. 2004); Dong Y et al.Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles for oraldelivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R.et al (Improved body distribution of 14C-labelled AZT bound tonanoparticles in rats determined by radioluminography. J Drug Target5:171.1998); Sakuma S R et al (Mucoadhesion of polystyrene nanoparticleshaving surface hydrophilic polymeric chains in the gastrointestinaltract. Int J Pharm 177:161. 1999); Virovic L et al. Novel deliverymethods for treatment of viral hepatitis: an update. Expert Opin DrugDeliv 2:707.2005); and Zimmermann E et al, Electrolyte- andpH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions inartificial gastrointestinal media. Eur J Pharm Biopharm 52:203. 2001).Each method represents a separate embodiment of the invention.

In one embodiment, mRNA is encapsulated in nanoparticles to improveefficiency of delivery and expression of vmRNA. Nanoparticle packaginginvolves condensing and encapsulating RNA into particles that aresmaller than the pore of the nuclear membrane, using chemicals includingpoly-L-lysine and polyethylene glycol. In one embodiment, RNA ispackaged into one of four nanoparticle formulations (PEI, PLL, PAE, andCK₃₀PEG_(10k)).

Lipid Nanoparticles

In one embodiment, nanoparticles used in the compositions and methods ofthe present invention comprise lipid nanoparticles as described inCullis, P., & Hope, M. (n.d.). Lipid Nanoparticle Systems for EnablingGene Therapies. Molecular therapy., 25(7), which is incorporated byreference herein in its entirety.

In one embodiment, delivery of nucleoside-modified RNA comprises anysuitable delivery method, including exemplary RNA transfection methodsdescribed elsewhere herein. In certain embodiments, delivery of anucleoside-modified RNA to a subject comprises mixing thenucleoside-modified RNA with a transfection reagent prior to the step ofcontacting. In another embodiment, a method of present invention furthercomprises administering nucleoside-modified RNA together with thetransfection reagent. In another embodiment, the transfection reagent isa cationic lipid reagent.

In another embodiment, the transfection reagent is a lipid-basedtransfection reagent. In another embodiment, the transfection reagent isa protein-based transfection reagent. In another embodiment, thetransfection reagent is a polyethyleneimine based transfection reagent.In another embodiment, the transfection reagent is calcium phosphate. Inanother embodiment, the transfection reagent is Lipofectin®,Lipofectamine®, or TransIT®. In another embodiment, the transfectionreagent is any other transfection reagent known in the art.

In another embodiment, the transfection reagent forms a liposome.

Liposomes, in another embodiment, increase intracellular stability,increase uptake efficiency and improve biological activity. In anotherembodiment, liposomes are hollow spherical vesicles composed of lipidsarranged in a similar fashion as those lipids which make up the cellmembrane. They have, in another embodiment, an internal aqueous spacefor entrapping water-soluble compounds and range in size from 0.05 toseveral microns in diameter. In another embodiment, liposomes candeliver RNA to cells in a biologically active form.

In one embodiment, the composition comprises a lipid nanoparticle (LNP)and one or more nucleic acid molecules described herein. For example, inone embodiment, the composition comprises an LNP and one or morenucleoside-modified RNA molecules encoding one or more antigens,adjuvants, or a combination thereof.

The term “lipid nanoparticle” refers to a particle having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includesone or more lipids, for example a lipid of Formula (I), (II) or (III),as described in WO2016176330A1, which is incorporated by referenceherein in its entirety.

In some embodiments, lipid nanoparticles are included in a formulationcomprising a nucleoside-modified RNA as described herein. In someembodiments, such lipid nanoparticles comprise a cationic lipid and oneor more excipient selected from neutral lipids, charged lipids, steroidsand polymer conjugated lipids (e.g., a pegylated lipid such as apegylated lipid of structure (IV), such as compound IVa). In someembodiments, the nucleoside-modified RNA is encapsulated in the lipidportion of the lipid nanoparticle or an aqueous space enveloped by someor all of the lipid portion of the lipid nanoparticle, therebyprotecting it from enzymatic degradation or other undesirable effectsinduced by the mechanisms of the host organism or cells e.g. an adverseimmune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, thenucleoside-modified RNA, when present in the lipid nanoparticles, isresistant in aqueous solution to degradation with a nuclease.

The LNP may comprise any lipid capable of forming a particle to whichthe one or more nucleic acid molecules are attached, or in which the oneor more nucleic acid molecules are encapsulated. The term “lipid” refersto a group of organic compounds that are derivatives of fatty acids(e.g., esters) and are generally characterized by being insoluble inwater but soluble in many organic solvents. Lipids are usually dividedin at least three classes: (1) “simple lipids” which include fats andoils as well as waxes; (2) “compound lipids” which include phospholipidsand glycolipids; and (3) “derived lipids” such as steroids.

In one embodiment, the LNP comprises one or more cationic lipids, andone or more stabilizing lipids. Stabilizing lipids include neutrallipids and pegylated lipids.

In one embodiment, the LNP comprises a cationic lipid. As used herein,the term “cationic lipid” refers to a lipid that is cationic or becomescationic (protonated) as the pH is lowered below the pK of the ionizablegroup of the lipid, but is progressively more neutral at higher pHvalues. At pH values below the pK, the lipid is then able to associatewith negatively charged nucleic acids. In certain embodiments, thecationic lipid comprises a zwitterionic lipid that assumes a positivecharge on pH decrease.

In certain embodiments, the cationic lipid comprises any of a number oflipid species which carry a net positive charge at a selective pH, suchas physiological pH. Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi),N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxy spermine (DOGS),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE). Additionally, a number of commercial preparations ofcationic lipids are available which can be used in the presentinvention. These include, for example, LIPOFECTIN® (commerciallyavailable cationic liposomes comprising DOTMA and1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, GrandIsland, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomescomprisingN-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.). The following lipids are cationic and have apositive charge at below physiological pH:

DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

In one embodiment, the cationic lipid is an amino lipid. Suitable aminolipids useful in the invention include those described in WO2012/016184, incorporated herein by reference in its entirety.Representative amino lipids include, but are not limited to,1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

In certain embodiments, the cationic lipid is present in the LNP in anamount from about 30 to about 95 mole percent. In one embodiment, thecationic lipid is present in the LNP in an amount from about 30 to about70 mole percent. In one embodiment, the cationic lipid is present in theLNP in an amount from about 40 to about 60 mole percent. In oneembodiment, the cationic lipid is present in the LNP in an amount ofabout 50 mole percent. In one embodiment, the LNP comprises onlycationic lipids. In certain embodiments, the L P comprises one or moreadditional lipids which stabilize the formation of particles duringtheir formation.

Suitable stabilizing lipids include neutral lipids and anionic lipids.

The term “neutral lipid” refers to any one of a number of lipid speciesthat exist in either an uncharged or neutral zwitterionic form atphysiological pH.

Representative neutral lipids include diacylphosphatidylcholines,diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

Exemplary neutral lipids include, for example,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl-phosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

In some embodiments, the LNPs comprise a neutral lipid selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, themolar ratio of the cationic lipid (e.g., lipid of Formula (I)) to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the LNPs further comprise a steroid or steroidanalogue.

In certain embodiments, the steroid or steroid analogue is cholesterol.In some of these embodiments, the molar ratio of the cationic lipid(e.g., lipid of Formula (I)) to cholesterol ranges from about 2:1 to1:1.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include phosphatidylglycerol,cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,N-dodecanoylphosphatidylethanolamines,N-succinylphosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

In certain embodiments, the LNP comprises glycolipids (e.g.,monosialoganglioside GMi). In certain embodiments, the LNP comprises asterol, such as cholesterol.

In some embodiments, the LNPs comprise a polymer conjugated lipid. Theterm “polymer conjugated lipid” refers to a molecule comprising both alipid portion and a polymer portion. An example of a polymer conjugatedlipid is a pegylated lipid. The term “pegylated lipid” refers to amolecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG)and the like.

In certain embodiments, the LNP comprises an additional,stabilizing-lipid which is a polyethylene glycol-lipid (pegylatedlipid). Suitable polyethylene glycol-lipids include PEG-modifiedphosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modifiedceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols.

Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA,and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid isN-[(methoxy poly(ethyleneglycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). Inone embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In otherembodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) suchas 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG),a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-0-(co-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asQ-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the cationic lipid to thepegylated lipid ranges from about 100:1 to about 25:1.

In certain embodiments, the additional lipid is present in the LNP in anamount from about 1 to about 10 mole percent. In one embodiment, theadditional lipid is present in the LNP in an amount from about 1 toabout 5 mole percent. In one embodiment, the additional lipid is presentin the LNP in about 1 mole percent or about 1.5 mole percent.

In certain embodiments, the LNP comprises one or more targeting moietieswhich are capable of targeting the LNP to a cell or cell population. Forexample, in one embodiment, the targeting moiety is a ligand whichdirects the LNP to a receptor found on a cell surface.

In certain embodiments, the LNP comprises one or more internalizationdomains. For example, in one embodiment, the LNP comprises one or moredomains which bind to a cell to induce the internalization of the LNP.For example, in one embodiment, the one or more internalization domainsbind to a receptor found on a cell surface to induce receptor-mediateduptake of the LNP. In certain embodiments, the LNP is capable of bindinga biomolecule in vivo, where the LNP-bound biomolecule can then berecognized by a cell-surface receptor to induce internalization. Forexample, in one embodiment, the LNP binds systemic ApoE, which leads tothe uptake of the LNP and associated cargo.

Other exemplary LNPs and their manufacture are described in the art, forexample in WO2016176330A1, U.S. Patent Application Publication No.US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176;Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, MolTher, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C NanomaterInterfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer.,131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37;Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Muiet al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, MolTher., 21(8): 1570-1578; and Tarn et al., 2013, Nanomedicine, 9(5):665-74, each of which are incorporated by reference in their entirety.

In another embodiment, methods of the present invention compriseadministering a modified mRNAs encoding HSV glycoprotein and apharmaceutically acceptable carrier or diluent. In other embodiments,pharmaceutically acceptable carriers for liquid formulations may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil.

As used herein “pharmaceutically acceptable carriers or diluents” arewell known to those skilled in the art.

In another embodiment, the pharmaceutical compositions provided hereinare controlled-release compositions, i.e. compositions in which thecompound is released over a period of time after administration.Controlled- or sustained-release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). In anotherembodiment, the composition is an immediate-release composition, i.e. acomposition in which the entire compound is released immediately afteradministration.

Each of the additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a kit comprising areagent utilized in performing a method of the present invention. Inanother embodiment, the present invention provides a kit comprising acomposition, tool, or instrument of the present invention.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXPERIMENTAL DETAILS SECTION Example 1: Materials and ExperimentalMethods

Modified mRNA expressing HSV-2 glycoproteins C, D and E (gC2/gD2/gE2)ectodomains. Modified mRNA (encoding gC2 (SEQ ID NO: 10), encoding gD2(SEQ ID NO: 4), and encoding gE2 (SEQ ID NO: 16)) was prepared based onthe DNA coding sequences that encode HSV-2 glycoprotein C (gC2) aminoacids 27-426 from HSV-2 strain 333 (SEQ ID NO: 11), glycoprotein D (gD2)amino acids 26-331 from HSV-2 strain 333 (SEQ ID NO: 5), andglycoprotein E (gE2) amino acids 24-405 from HSV-2 strain 2.12 (SEQ IDNO: 17).

The modified mRNA was incorporated into liposomal nanoparticles (LNP) byAcuitas Therapeutics to prepare the following immunogens: (a) Poly CmRNA in LNP; (b) gC2 modified mRNA in LNP; (c) gD2 modified mRNA in LNP;(d) gE2 modified mRNA in LNP; (e) gC2 & gD2 & gE2 modified mRNA in LNP.

Immunization groups were as follows:

-   -   a) Controls (Poly C group): 10 μg Poly C mRNA/LNP divided into 4        aliquots and administered at 4 separate sites.    -   b) gD2 alone (gD2 group): 10 μg gD2 mRNA/LNP divided into 4        aliquots and administered at 4 separate sites.    -   c) Individual trivalent (Trivalent-I group): 10 μg gC2 mRNA/LNP,        10 μg gD2 mRNA/LNP, 10 μg gE2 mRNA/LNP each divided into 2        aliquots and each given at 2 sites.    -   d) Combined trivalent (Trivalent-C group): 10 μg gC2 mRNA & 10        μg gD2 mRNA & 10 μg gE2 mRNA combined into LNP and divided into        4 aliquots and given at 4 sites.

Experimental Procedures. Hair was removed from the back of 6-8 week oldBALB/c mice using an electric razor and Nair. Mice were bled prior tothe first and second immunization and prior to intravaginal challenge.Two immunizations were performed intradermally at 28-day intervals.Intradermal immunizations were performed on the denuded backs. Five micethat received the trivalent vaccine at individual sites (group c above)were sacrificed 14 days after the second immunization. Spleens wereharvested for CD4⁺ and CD8⁺ T cell responses to gC2, gD2 and gE2 subunitantigens or to 15 amino acid peptides each with 11 overlapping aminoacids. Twenty-eight days after the second immunization, mice weretreated subcutaneously with 2 mg Depo-Provera and 5 days later infectedintravaginally with 5×10³ PFU HSV-2 strain MS (˜400 LD₅₀). On days 2 and4 post-challenge, vaginal swabs were obtained for virus cultures. On day4 post-challenge, some mice in each vaccine group were sacrificed anddorsal root ganglia (DRG) excised for HSV-2 DNA qPCR. The remaininganimals were evaluated for weight loss and hind limb weakness for 10days while survival and genital disease were monitored for 28 days.

Example 2: Characterization of Translational Products Produced by Gc2,Gd2, And Ge2 Modified mRNA

The ability of modified mRNA to express proteins of the expectedmolecular weight when transfected into mammalian cells was verified. 0.1μm of gC2-, gD2-, or gE2-modified mRNA was transfected into 293T cellsusing TransIT-mRNA (Mirus Bio LLC) for the transfection. Eighteen hourslater, cells were harvested and extracts prepared for Western blots. ThemRNAs were designed to express the ectodomains of gC2, gD2 and gE2(labeled mRNA-ecto). As controls for the expected molecular weights,purified baculovirus proteins gC2, gD2, and gE2 expressing the sameamino acids as the mRNA constructs (labeled Bac-ecto) were used (FIGS.1A-C).

Conclusion: When transfected into mammalian cells, modified mRNAencoding the ectodomains of HSV-2 gC2 (FIG. 1A), gD2 (FIG. 1B) and gE2(FIG. 1C) produced proteins of the appropriate molecular weights thatreacted with antibodies to the glycoproteins on Western blot.

Example 3: Elisa Antibody Responses in Subjects Immunized with gD2 orTrivalent Modified mRNA Vaccines

ELISA endpoint titers were evaluated on sera taken 28 days after thefirst and second immunizations. Immunization groups were as follows:Poly C (10 μg Poly C mRNA/LNP divided into 4 aliquots and administeredat 4 separate sites) (control); gD2 (10 μg gD2 mRNA/LNP divided into 4aliquots and administered at 4 separate sites); Trivalent-I (10 μg gC2mRNA/LNP, 10 μg gD2 mRNA/LNP, 10 μg gE2 mRNA/LNP each divided into 2aliquots and each given at 2 sites); and Trivalent-C (10 μg gC2 mRNA &10 μg gD2 mRNA & 10 μg gE2 mRNA combined into LNP and divided into 4aliquots and given at 4 sites).

Four animals were evaluated in each group. High ELISA titers to eachimmunogen were obtained after the first immunization (marked as romannumeral I; FIGS. 2A-C), and the titers were boosted even higher afterthe second immunization (marked as roman numeral II; FIGS. 2A-C).Immunization with gD2 modified mRNA vaccine selectively inducedextremely high titers of ELISA antibodies to gD2 (FIG. 2B), whileimmunization with the trivalent modified mRNA vaccines induced extremelyhigh titers of ELISA antibodies to gC2 (FIG. 2A) and gD2 (FIG. 2B) andhigh titers to gE2 (FIG. 2C). In all non-control groups, the secondimmunization significantly boosted the ELISA titers compared to thefirst. The differences between the titers in the second vs the firstimmunizations were significant, p<0.05 (t-tests, comparing the antibodytiters after the first and second immunization).

Conclusion: The gD2 mRNA and gC2, gD2 and gE2 mRNA immunogens inducedextremely high titers of ELISA antibodies after the first immunizationthat were significantly boosted after the second immunization.

Example 4: Balanced T_(H)1 and T_(H)2 Igg Isotypes Produced By ModifiedmRNA Immunization

The ability of mRNA immunizations to stimulate predominantly a T_(H)1 orT_(H)2 immune response was tested by determining whether IgG1 (T_(H)2)or IgG2a (T_(H)1) antibodies are produced. ELISA was performed on platescoated with all three antigens, gC2, gD2 and gE2. Serum obtained afterthe first or second immunization was added to the antigen-coated plates,and IgG1 or IgG2a was detected using HRP anti-mouse IgG1 or IgG2a. IgG1(FIG. 3A) and IgG2a (FIG. 3B) titers were significantly elevated afterimmunization with gD2 and the trivalent modified mRNA vaccines. Further,the IgG1 (FIG. 3A) or IgG2a (FIG. 3B) titers were significantly higherafter the second modified mRNA immunization compared to the first,p<0.05 (t tests).

Conclusion: The results demonstrate high titers of antibodies areproduced to both IgG1 and IgG2a isotypes, indicating a balanced T_(H)1and T_(H)2 response to immunization with modified gC2, gD2 and gE2 mRNA.

Example 5: High Neutralizing Antibody Titers After Modified mRNAImmunization

Serum was obtained 28 days after the second immunization, andneutralizing antibody titers were determined using serial 2-folddilutions of serum, starting at a 1:25 dilution and 10% human serum as asource of complement. The human serum was obtained from an individualseronegative for HSV-1 and HSV-2. The modified mRNA groups were eachsignificantly different from the poly C controls (p<0.001; FIG. 4 ).While each of the mRNA groups was not significantly different from oneanother, the trivalent vaccine given as a combined immunogen(Trivalent-C) performed the best of the three mRNA groups (FIG. 4 ).

Conclusion: Each of the modified mRNA groups produced extremely hightiters of neutralizing antibodies in the presence of 10% humancomplement.

Example 6: CD4⁺ AND CD8⁺ T Cell Responses in Splenocytes After ModifiedmRNA Immunization

Five animals from the trivalent modified mRNA group that were immunizedwith each glycoprotein mRNA at a separate site (Trivalent-I group) wereeuthanized 14 days after the second immunization. Splenocytes wereprepared for T cell assays. Splenocytes were stimulated withglycoprotein subunit antigens prepared in baculovirus or 15 amino acidpeptides containing 11 overlapping amino acids. The CD4⁺ and CD8⁺ T cellresponses are shown in FIGS. 5A-5B and FIGS. 6A-6B, respectively.

CD4⁺ T cells: The modified mRNA-expressed gC2, gD2, and gE2 subunitantigens each stimulated polyfunctional CD4⁺ T cell responses (FIGS.5A-5B). Splenocytes harvested from immunized subjects and thenstimulated with subunit antigen glycoproteins increased polyfunctionalCD4⁺ T cell responses (FIG. 5A). Splenocytes harvested from immunizedsubjects and then stimulated with 15 amino acid overlapping peptidesincreased polyfunctional CD4⁺ T cell responses and IFNγ responses (FIG.5B). CD8⁺ T cells: Only gE peptide pool 2 stimulated a significant IFNγCD8⁺ T cell response (FIG. 6B).

Example 7: Survival, Weight Loss and Neurological Signs After ModifiedmRNA Immunization and Intravaginal Challenge

Thirty-three days after the second immunization, animals were inoculatedintravaginally with 5×10³ PFU of HSV-2 strain MS (˜400 LD₅₀). Animalswere observed daily for survival, neurological signs consisting of hindlimb weakness or paralysis and hunched gait, and for weight loss orgain. All animals in the poly C control group died, while all animals inthe gD2 alone, trivalent given individually (labeled Trivalent-I) ortrivalent given combined (labeled Trivalent-C) survived (FIG. 7A;p=0.002 by Log-rank (Mantel-Cox) comparing the three mRNA/LNP groupswith poly C controls). FIG. 7B demonstrates that administration of themodified mRNA vaccine twice at 28 day intervals and challengedintravaginally with HSV-2 does not result in neurological signs orweight loss. Control subjects that were not administered the vaccine andwere and challenged intravaginally with HSV-2 showed weight loss andneurological signs.

Each of the mRNA/LNP groups significantly outperformed the controlgroup. All mice immunized with the modified mRNA survived and showed noevidence of weight loss, neurological disease or genital lesions afterintravaginal challenge with ˜400 LD₅₀ of HSV-2.

Example 8: HSV-2 Vaginal Titers After Modified mRNA Immunization andIntravaginal Challenge

Vaginal swabs were obtained from 10 animals per group on days 2 and 4post challenge and cultured for replication competent HSV-2 virus.Results are shown in FIGS. 8A-8B. 9/10 animals in the poly C group hadpositive cultures on days 2 (FIG. 8A) and 4 (FIG. 8B) compared with 3/10in the gD2 group and 0/10 in the trivalent-I or trivalent-C groups (Pvalues by Fisher Exact test were not significant comparing trivalentgroups to gD2 alone; p<0.001 comparing trivalent-I or trivalent-C withpoly C; p=0.02 comparing gD2 alone with poly C).

Each of the mRNA/LNP groups significantly outperformed the Poly Ccontrol group. Remarkably, day 2 and day 4 vaginal titers afterchallenge were negative in mice immunized with the trivalent mRNAwhether given at separate sites or as a combined immunization. Nosignificant differences were detected comparing either trivalent groupwith gD2 alone, although both trivalent groups outperformed the gD2alone group, as 3 of 10 mice in the gD2 group had virus isolated fromvaginal swabs.

Example 9: Genital Disease After Modified mRNA Immunization andIntravaginal Challenge

Animals were monitored daily for genital disease for 28 days postchallenge. A score of 0 was assigned for no disease, and 1 point eachwas assigned for hair loss around the anal or genital orifices, genitalerythema, genital exudate, and necrosis of genital tissues (FIG. 9 ).

No animal in the gD2 or trivalent mRNA/LNP groups developed genitaldisease, which was significantly different than the poly C controls(p<0.001, one-way ANOVA by Kruskal-Wallis test followed by Dunn'smultiple comparisons for significance).

Example 10: HSV-2 DNA in Dorsal Root Ganglia After Modified mRNAImmunization and Intravaginal Challenge

Five animals per group were euthanized at 4 days post challenge, exceptfor the trivalent-combined group in which four animals were euthanized.Dorsal root ganglia (DRG) were harvested for HSV-2 DNA quantitation byqPCR to detect the Us9 gene. All five animals in the poly C group hadHSV-2 DNA detected in the DRG, while 2/5 animals in the gD mRNA, 1/5 inthe trivalent mRNA at individual sites, and 1/4 trivalent mRNA given atthe same site were positive for HSV-2 DNA (FIG. 10 ; Mann-Whitney test:gD2 compared with poly C, p=0.03; trivalent at different sites comparedwith poly C, p<0.01; trivalent at same site compared with poly C,p=0.14). The difference between the modified mRNA immunized groups wasnot significant.

Conclusion: Dorsal root ganglia were negative for HSV-2 DNA on day 4after infection in 75% to 80% of animals immunized with gD2 alone or thetrivalent vaccine. The trivalent mRNA group at different sites and thegD2 mRNA group significantly outperformed the poly C mRNA control group,while the trivalent mRNA group with all glycoproteins given together didnot differ significantly from the poly C group, likely because of thesmaller sample size in the trivalent-combined group.

Summary

Modified mRNA vaccines expressing gD2 alone or gC2, gD2 and gE2 providedoutstanding protection against HSV-2 genital challenge. The expressionof the three proteins slightly outperformed gD2 based on day 2 and day 4titers after challenge and the lower number of animals with HSV-2 DNAdetected in DRG on day 4.

Example 11: T Follicular Helper (Tfh) Cell and Germinal Center B CellResponses in Immunized Mice

BALB/c female mice were left un-immunized as naïve control animals orimmunized intradermally twice at 28 day intervals with poly C mRNA-LNPor trivalent modified mRNA-LNP. The poly C mRNA controls received 10 μgPoly C mRNA-LNP divided into 4 aliquots and administered at 4 separatesites. The trivalent modified mRNA group received 10 μg gC2 mRNA-LNP, 10μg gD2 mRNA-LNP, and 10 μg gE2 mRNA-LNP each divided into 2 aliquots andeach given at 2 sites. Two weeks after the second immunization, spleenswere harvested from 5 animals per group and flow cytometry performed todetect T follicular helper (Tfh) cells (FIG. 11A; *p<0.05) and germinalcenter B cell responses (FIG. 11B; *p<0.05).

Conclusion: The trivalent mRNA-LNP vaccine induced a potent Tfh andgerminal center B cell response and significantly outperformed the polyC control immunization (p<0.05) and the naïve group (p<0.05) for bothTfh and germinal center B cell responses. These immune responses suggestthat the trivalent modified mRNA-LNP vaccine will likely induce adurable antibody response.

Example 12: Vaginal IgG Responses to Modified mRNA Immunization in Mice

BALB/c mice were immunized intradermally twice at 28 day intervals with10 μg of ploy C mRNA-LNP, 10 μg gD2 mRNA-LNP or 10 μg each of gC2, gD2,gE trivalent modified mRNA-LNP. The trivalent mRNA was combined andadministered as 10 μg gC2 mRNA & 10 μg gD2 mRNA & 10 μg gE2 mRNAcombined into LNP and divided into 4 aliquots and given at 4 sites. Onemonth after the second immunization, 60 μl of media was introduced inthe vaginal cavity and retrieved. IgG titers to gC2 (FIG. 12A), gD2(FIG. 12B), and gE2 (FIG. 12C) were determined at a 1:50 dilution of thevaginal wash fluids by ELISA (FIGS. 12A-12C, n=10 mice in the poly Cgroup, n=10 in the gD2 mRNA group and n=25 in the trivalent mRNA group;***p<0.001; **p<0.01).

Conclusion: The trivalent mRNA produced a robust vaginal IgG response togC2 (FIG. 12A) and gD2 (FIG. 12B) and a more moderate response to gE2(FIG. 12C). The gD2 ELISA titers were higher in mice immunized with themodified trivalent mRNA vaccine compared to mice immunized with themodified gD2 mRNA vaccine (FIG. 12B).

Example 13: Antibodies to gC2 Produced By Trivalent mRNA Immunization ofMice Block Immune Evasion Domains on gC2

BALB/c mice were left unimmunized as a source of non-immune IgG, orimmunized intradermally with poly C mRNA-LNP or trivalent mRNA-LNP. Thepoly C mRNA controls received 10 μg poly C mRNA-LNP divided into 4aliquots and administered at 4 separate sites. The gD2 mRNA groupreceived 10 μg gD2 mRNA-LNP administered as described for the poly CmRNA-LNP. The trivalent modified mRNA group received 10 μg gC2 mRNA-LNP,10 μg gD2 mRNA-LNP, and 10 μg gE2 mRNA-LNP combined into one LNP anddivided into 4 aliquots and given at 4 sites. There were 10 mice in eachgroup. Sera from the 10 mice were pooled and IgG was purified. The IgGwas evaluated at 12 μg/200 μl for its ability to block complementcomponent C3b binding to gC2. This blocking assay is used to assesswhether antibodies produced by immunization block the immune evasionproperties of gC2. Non-immune murine IgG, IgG from the poly C mRNAgroup, and IgG from the gD2 mRNA group each failed to block gC2 bindingto C3b. In contrast, IgG from trivalent mRNA-immunized animals totallyblocked the interaction between gC2 and C3b (FIG. 13 , ****p<0.0001).

Conclusions: The trivalent mRNA vaccine produces antibodies that blockimmune evasion domains on gC2 as determined by blocking the interactionbetween gC2 and C3b.

Example 14: Intravaginal Infection of Mice at a Higher Inoculum Titer OfHSV-2 After Modified mRNA Vaccination

BALB/c mice (n=5) were immunized with the trivalent modified mRNA using10 μg gC2 mRNA-LNP, 10 μg gD2 mRNA-LNP, 10 μg gE2 mRNA-LNP each dividedinto 2 aliquots and each given individually at 2 sites. One month afterthe second immunization, mice were treated with medroxyprogesterone andfive days later infected intravaginally with 5×10⁴ PFU HSV-2 strain MS(2,000 LD₅₀). Animals were followed for 28 days and evaluated for death,genital disease, vaginal viral titers 2 and 4 days after infection anddorsal root ganglia (DRG) HSV-2 DNA copy number 28 days after infection.No mouse immunized with the trivalent mRNA-LNP vaccine died, had genitaldisease, had any virus detected on day 2 or 4 post-infection or hadHSV-2 DNA detected in DRG (Table 1).

TABLE 1 Trivalent mRNA-LNP immunized mice challenged with HSV-2 strainMS (2,000 LD₅₀) Disease parameters Mice % Protection Death 0/5 100Genital disease 0/5 100 Vaginal viral titers day 2 0/5 100 Vaginal viraltiters day 4 0/5 100 HSV-2 DNA copies in DRG 0/5 100

Conclusions: Mice were infected with HSV-2 at a dose that was 10-foldhigher than used in earlier experiments described herein (FIGS. 7A-10 ).Protection of the mice remained outstanding even at this higher titerchallenge. We achieved sterilizing immunity in all five mice asdetermined by no deaths, no genital disease, negative vaginal virustiters on days 2 and 4 post-infection and no HSV-2 DNA in thelumbosacral DRG on day 28 (Table 1).

Example 15: Evaluation of the Intramuscular Route of Modified mRNAImmunization in Mice

BALB/c mice were immunized intramuscularly with poly C mRNA-LNP as acontrol (15/group) or with trivalent mRNA containing 10 μg each of gC2,gD2 and gE2 mRNA-LNP (20/group). All poly C control animals died by day12, while all animals in the trivalent mRNA group survived (FIG. 14A).No weight loss occurred in the trivalent mRNA group, while the poly Ccontrol animals lost >15% of body weight (FIG. 14B). The poly C groupdeveloped extensive genital disease, while the trivalent mRNA animalshad no genital disease (FIG. 14C). DRG were harvested from nine poly Canimals at the time of euthanasia between days 7 and 12 post-infectionor at the end of the experiment on day 28 in the trivalent mRNA group.All animals in the poly C group had HSV-2 DNA detected in DRG, whilenone were positive for HSV-2 DNA in trivalent mRNA group (FIG. 14D). Day2 (FIG. 14E) and Day 4 (FIG. 14F) vaginal viral cultures were positivein all 15 animals in the poly C group, while cultures were negative inall 20 animals in the trivalent mRNA group. Differences between poly Cand trivalent groups are significant, p<0.001 for each figure (FIGS.14A-14F).

Conclusions: Trivalent modified mRNA-LNP provides outstanding protectionin mice when administered intramuscularly. We reported comparablefindings above when mice were immunized intradermally. Overall, we havenow evaluated 64 mice that were immunized with trivalent mRNA at 10 μgof each immunogen given either intradermally (FIGS. 7A-10 ) orintramuscularly (FIGS. 14A-14F). We have achieved sterilizing immunityin 63/64 (98%) mice based on no death, no genital disease, no weightloss, negative day 2 and 4 vaginal titers and negative HSV-2 DNA in DRG.

Example 16: Summary Comparison of Immunization with Trivalent mRNA-LNPAnd Trivalent Subunit Antigen CPG/ALUM in BALB/c Mice

The results presented in Table 2 hereinbelow represent a summary of allthe results in BALB/c mice that were immunized either intradermally orintramuscularly with trivalent mRNA containing 10 μg each of gC2, gD2and gE2 mRNA-LNP (total 64 mice studied). We show a comparison with theresults obtained in BALB/c mice that were immunized with 5 μg each ofbac-gC2(27-426t) containing gC2 amino acids 27-426 from HSV-2 strain333, bac-gD2(306t) containing gD2 amino acids 26-331 from HSV-1 strain333, and bac-gE2(24-405t) containing gE2 amino acids 24-405 from HSV-2strain 2.12. The gC2, gD2, gE2 subunit antigens were mixed with 150 μgCpG and 25 μg alum/per μg protein as adjuvants and administeredintramuscularly. Mice were immunized twice at 28-day intervals withtrivalent mRNA-LNP and three times at 14-day intervals with subunitantigens, as we have done in prior experiments. The mRNA and subunitantigen experiments were performed at the same time. The resultssummarized in Table 2 demonstrate significant superiority of thetrivalent mRNA-LNP vaccine over the trivalent subunit antigen vaccine inmany immune response parameters, and most importantly in vaccineefficacy. The trivalent mRNA-LNP vaccine achieved sterilizing immunityin 63/64 (98%) of mice compared to 15/20 (75%) in the subunit antigengroup.

TABLE 2 Comparisons of immunization with trivalent mRNA-LNP or trivalentsubunit antigen CpG/alum in BALB/c mice. Trivalent subunit ComparisonTrivalent mRNA antigen P value Serum IgG ELISA gC2 1:256,000 1:32,000 p< 0.001 gD2 1:512,000 1:128,000 p < 0.01 gE2 1:64,000 1:16,000 p < 0.05Vaginal fluid IgG ELISA OD at 405 nm tested at 1:50 dilution gC2 1.6 OD0.6 OD p < 0.001 gD2 1.5 OD 1.0 OD p < 0.05 gE2 0.5 OD 0.25 OD p < 0.01Serum neutralizing antibody Against HSV-2 1:4,800 1:1,600 p < 0.01Against HSV-1 1:6,400 1:4,000 p = NS* Blocking C3b Total blocking Totalblocking p = NS binding to gC2 CD4⁺ T cell Significant SignificantTrivalent responses responses for gC2, response only mRNA more gD2 andgE2 for gD2 potent CD8⁺ T cell Significant No significant Trivalentresponses response for gE2 responses mRNA more potent Achieving 63/64(98%) mice 15/20 (75%) p < 0.01 sterilizing mice immunity^(#) *NS, notsignificant; ^(#)Sterilizing immunity defined as no death, no genitaldisease, no weight loss, and no evidence of subclinical infection asmeasured by day 2 and day 4 vaginal cultures post-infection and HSV-2DNA in dorsal root ganglia on day 4 or day 28 post-infection.

Example 17: Evaluation of the Trivalent mRNA-LNP Vaccine in Guinea Pigs

Hartley Strain female guinea pigs were left unimmunized and uninfected(naive group, n=10), immunized three times intradermally at monthlyintervals with 20 μg poly C mRNA-LNP (n=10) or with 20 μg each of gC2,gD2, gE modified mRNA-LNP (n=10). One month after the finalimmunization, animals in the poly C and trivalent mRNA groups wereinfected intravaginally with 5×10⁵ PFU of HSV-2 strain MS (50 LD₅₀).Animals were observed for death, genital lesions during the acute phaseof infection (days 1-14) and genital lesions during the recurrent phaseof infection (days 15-60). In the poly C control group, 7/10 animalsdied or were humanly euthanized between days 7 and 20 post-infection,while no animal in the trivalent group and no naïve (uninfected) animaldied (FIG. 15A). The poly C group had genital lesions on a mean of 6.4days during the acute phase of infection with 9/10 animals developingacute genital disease, while no animal in the trivalent group or naïve(uninfected) group developed acute genital disease (FIG. 15B). The polyC animals had genital lesions on a mean of 3.7 days during the recurrentphase of infection from days 15-60, with 2/3 animals developingrecurrent genital lesions (FIG. 15C). In contrast, the trivalentimmunized guinea pigs and the naïve (uninfected) animals had norecurrent genital lesions (FIG. 15C).

Conclusions: Trivalent modified mRNA-LNP provided outstanding protectionagainst acute and recurrent genital lesions in guinea pigs.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to the precise embodiments, and that various changes andmodifications may be effected therein by those skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

All patent documents and references cited herein are incorporated byreference as if fully set forth.

What is claimed is:
 1. A composition comprising (a) RNA encoding theectodomain of Herpes Simplex Virus (HSV) glycoprotein D (gD), whereinthe ectodomain consists of a sequence that is at least 98% identical toSEQ ID NO: 5, (b) RNA encoding the ectodomain of HSV glycoprotein C(gC), wherein the ectodomain consists of a sequence that is at least 98%identical to SEQ ID NO: 11, and (c) RNA encoding the ectodomain of HSVglycoprotein E (gE), wherein the ectodomain consists of a sequence thatis at least 98% identical to SEQ ID NO: 17, wherein one or more of saidRNAs is a nucleoside modified RNA.
 2. The composition of claim 1,wherein said nucleoside modified RNA comprises one or more pseudouridineresidues.
 3. The composition of claim 2, wherein said one or morepseudouridine residues comprise m1Ψ (1-methylpseudouridine), m1acp3Ψ(1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, Ψm(2′-O-methylpseudouridine, m5D (5-methyldihydrouridine), m3Ψ(3-methylpseudouridine), or any combination thereof.
 4. The compositionof claim 1, wherein said composition further comprises one or morenucleoside modified RNAs encoding a) HSV glycoprotein B (gB) orimmunogenic fragment thereof, b) HSV glycoprotein H (gH) or immunogenicfragment thereof, c) HSV glycoprotein L (gL) or immunogenic fragmentthereof, d) HSV glycoprotein I (gI) or immunogenic fragment thereof, ore) any combination thereof.
 5. The composition of claim 4, wherein oneor more of said further glycoproteins is from HSV-2.
 6. The compositionof claim 1, wherein one or more of said nucleoside modified RNAs furthercomprise a signal sequence.
 7. The composition of claim 1, where saidsignal sequence comprises: a) (SEQ ID NO: 19)AUGACCCGCCUGACCGUGCUGGCCCUGCUGGCCGGCCUGCUGGCCUCCUCC CGCGCC, b)(SEQ ID NO: 20) AUGCGCAUGCAGCUGCUGCUGCUGAUCGCCCUGUCCCUGGCCCUGGUGACCAACUCC, or c) (SEQ ID NO: 21)AUGGCCAUCUCCGGCGUGCCCGUGCUGGGCUUCUUCAUCAUCGCCGUGCUGAUGUCCGCCCAGGAGUCCUGGGCC.


8. The composition of claim 1, wherein one or more of said nucleosidemodified RNAs a) further comprise i) a poly-A tail; ii) an m7GpppG cap,3′-O-methyl-m7GpppG cap, or anti-reverse cap analog; iii) acap-independent translational enhancer; iv) 5′ and 3′ untranslatedregions that enhance translation; v) or a combination thereof; b) areencapsulated in a nanoparticle, lipid, polymer, cholesterol, or cellpenetrating peptide; c) or a combination thereof.
 9. The composition ofclaim 8, wherein said nanoparticle is a liposomal nanoparticle.
 10. Amethod of treating a Herpes Simplex Virus (HSV) infection orsuppressing, inhibiting, or reducing the incidence of an HSV infectionin a subject, the method comprising the step of administering to saidsubject (a) RNA encoding the ectodomain of Herpes Simplex Virus (HSV)glycoprotein D (gD), wherein the ectodomain consists of a sequence thatis at least 98% identical to SEQ ID NO: 5; (b) RNA encoding theectodomain of HSV glycoprotein C (gC), wherein the ectodomain consistsof a sequence that is at least 98% identical to SEQ ID NO: 11; and (c)RNA encoding the ectodomain of HSV glycoprotein E (gE), wherein theectodomain consists of a sequence that is at least 98% identical to SEQID NO: 17, wherein one or more of said RNAs is a nucleoside-modifiedRNA.
 11. The method of claim 10, wherein said HSV infection comprises anHSV-1 infection or an HSV-2 infection.
 12. The method of claim 10,wherein said HSV infection comprises a primary HSV infection; a flare,recurrence, or HSV labialis following a primary HSV infection; areactivation of a latent HSV infection; an HSV encephalitis; an HSVneonatal infection; a genital HSV infection; or an oral HSV infection;or a combination thereof.
 13. The method of claim 10, wherein theadministration step comprises intramuscular, subcutaneous, intradermal,intranasal, intravaginal, intrarectal, or topical administration. 14.The method of claim 10, wherein the administration step comprises a)administering a first composition comprising said RNA encoding theectodomain of HSV gD, b) administering a second composition comprisingsaid RNA encoding the ectodomain of HSV gC, and c) administering a thirdcomposition comprising said RNA encoding the ectodomain of HSV gE. 15.The method of claim 10, further comprising the step of administering tosaid subject a composition comprising a HSV glycoprotein selected fromgD, gC, and gE.
 16. A method of inducing an immune response in asubject, comprising the step of administering to said subject (a) RNAencoding the ectodomain of Herpes Simplex Virus (HSV) glycoprotein D(gD), wherein the ectodomain consists of a sequence that is at least 98%identical to SEQ ID NO: 5; (b) RNA encoding the ectodomain of HSVglycoprotein C (gC), wherein the ectodomain consists of a sequence thatis at least 98% identical to SEQ ID NO: 11; and (c) RNA encoding theectodomain of HSV glycoprotein E (gE), wherein the ectodomain consistsof a sequence that is at least 98% identical to SEQ ID NO: 17, whereinone or more of said RNAs is a nucleoside-modified RNA.
 17. The method ofclaim 16, wherein the administration step comprises intramuscular,subcutaneous, intradermal, intranasal, intravaginal, intrarectal, ortopical administration.
 18. The method of claim 16, wherein theadministration step comprises a) administering a first compositioncomprising said RNA encoding the ectodomain of HSV gD, b) administeringa second composition comprising said RNA encoding the ectodomain of HSVgC, and c) administering a third composition comprising said RNAencoding the ectodomain of HSV gE.
 19. The method of claim 16, furthercomprising the step of administering to said subject a compositioncomprising an HSV glycoprotein selected from gD, gC, and gE.
 20. Themethod of claim 16, wherein said immune response comprises a CD4 immuneresponse; a CD8 immune response; a T follicular helper cell immuneresponse; a germinal center B cell immune response; an IgG antibodyresponse to gC2, gD2, gE2, or combination thereof; or a combinationthereof.
 21. The composition of claim 1, wherein said RNA encoding theectodomain of HSV gD consists of SEQ ID NO: 22; wherein said RNAencoding the ectodomain of HSV gC consists of SEQ ID NO: 23, and whereinsaid RNA encoding the ectodomain of HSV gE consists of SEQ ID NO: 24.22. The method of claim 10, wherein said RNA encoding the ectodomain ofHSV gD consists of SEQ ID NO: 22; wherein said RNA encoding theectodomain of HSV gC consists of SEQ ID NO: 23, and wherein said RNAencoding the ectodomain of HSV gE consists of SEQ ID NO:
 24. 23. Thecomposition of claim 1, wherein two or more of said RNAs are nucleosidemodified RNAs.
 24. The composition of claim 1, wherein all three of saidRNAs are nucleoside modified RNAs.
 25. The method of claim 16, whereinsaid RNA encoding the ectodomain of HSV gD consists of SEQ ID NO: 22;wherein said RNA encoding the ectodomain of HSV gC consists of SEQ IDNO: 23, and wherein said RNA encoding the ectodomain of HSV gE consistsof SEQ ID NO: 24.